WO2020153815A1 - Novel kazachstania turicensis cau y1706 and composition using same - Google Patents

Novel kazachstania turicensis cau y1706 and composition using same Download PDF

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WO2020153815A1
WO2020153815A1 PCT/KR2020/001222 KR2020001222W WO2020153815A1 WO 2020153815 A1 WO2020153815 A1 WO 2020153815A1 KR 2020001222 W KR2020001222 W KR 2020001222W WO 2020153815 A1 WO2020153815 A1 WO 2020153815A1
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strain
culture
composition
disease
present
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PCT/KR2020/001222
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French (fr)
Korean (ko)
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김원용
김종화
김기영
렁라비칸자나순트리
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중앙대학교 산학협력단
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Priority to US17/425,079 priority Critical patent/US20220106559A1/en
Publication of WO2020153815A1 publication Critical patent/WO2020153815A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
    • A23L31/10—Yeasts or derivatives thereof
    • A23L31/15—Extracts
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9728—Fungi, e.g. yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/165—Yeast isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/14—Yeasts or derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/318—Foods, ingredients or supplements having a functional effect on health having an effect on skin health and hair or coat
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the present invention relates to a novel Kazaztania thuricensis CAU Y1706 and a composition using the same, and more specifically, the Kazaztania thuricensis CAU Y1706 (Accession No.: KCTC13794BP) strain, and the strain or a culture thereof are effective. It relates to a pharmaceutical composition, a food composition, a cosmetic composition, and a probiotic composition for the prevention or treatment of inflammatory diseases including as a component.
  • Inflammation is a continuous and complex reaction that destroys cells in the body.
  • Inflammatory reaction refers to a pathological condition formed by the invasion of external infectious agents (bacteria, fungi, viruses, and various allergens), and the damage site when an invasion that causes any organic changes to cells or tissues of these organisms is applied Is a defense reaction process of a living body that is trying to restore and regenerate.
  • an inflammatory reaction is a biological defense mechanism to regenerate damage due to invasion, which causes any organic changes in cells or tissues of a living body.
  • this reaction process local blood vessels, various tissue cells of body fluids, and immune cells act do. While the inflammatory reaction normally induced by external invading bacteria is a protective action to protect the living body, various diseases appear when abnormally excessive inflammatory reaction is induced.
  • Asthma one of chronic inflammatory diseases, is caused by airway hyperresponsiveness, deposition of inflammatory cells into the lung tissue, and overproduction of mucus, and cytokines including interleukin (IL)-4, IL-5, and IL-13.
  • Allergic asthma is induced by overexpression of chemokines such as cain, eotexin, and lentis.
  • B cells, mast cells and eosinophils cells produce various inflammation-inducing mediators.
  • Eosinophilic cells increase the amount of particulate proteins and create reactive oxygen species as the primary mediator of asthma development.
  • Allergy is a systemic or local disorder of a living body based on an immune response that is the generalization of a wide range of complex pathological phenomena. Allergies in the human body are classified into I, II, III, and IV types according to the immune mechanism, among which type I allergies belonging to the immediate hypersensitivity reaction occupy an important part in clinical practice, atopic dermatitis, allergic rhinitis, These include bronchial asthma, hyperthermia and pollinosis.
  • Asthma which is known as a representative disease of type I allergies, is an allergic disease.
  • Clinical symptoms such as wheezing, dyspnea, and cough caused by widespread narrowing of the airways can be reversibly improved naturally or by treatment.
  • the number of deaths from asthma is increasing worldwide, and 3-10% of developing countries suffer from asthma.
  • antigen-specific IgE binds to the IgE receptor on the surface of Langerhans cells, and is delivered to T cells on the surface of this antigen to activate T cells.
  • Th2 is activated in allergic skin diseases, especially atopic dermatitis, and cytokines such as IL-4, IL-5, IL-6, IL-8, IL-10 and IL-13 are secreted,
  • cytokines such as IL-4, IL-5, IL-6, IL-8, IL-10 and IL-13 are secreted
  • Kefir is a well-known probiotic food that is produced by symbiotic fermentation of lactic acid bacteria and yeast in milk.
  • Kazaztania Turisensis which belongs to kefir yeast, was previously known as saccharomyces turicensis and is isolated from various kefir grains (System. Apple.Microbiol. 22, 420-425 ( 1999).
  • the present inventors regulate the immune response, and the novel ability to modulate the immune response in the ovalbumin (OVA)-sensitized AD rat model and the ability to change the intestinal microflora is a new kazachstania turicensis.
  • OVA ovalbumin
  • the present invention was completed by separating CAU Y1706.
  • an object of the present invention is to provide a strain of Kazachstania turicensis CAU Y1706 (Accession No.: KCTC13794BP).
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases comprising the strain or a culture thereof as an active ingredient.
  • compositions for the prevention or treatment of inflammatory diseases comprising (essentially) the strain or a culture thereof as an active ingredient.
  • Another object of the present invention is to provide a food composition for preventing or improving inflammatory diseases comprising the strain or a culture thereof as an active ingredient.
  • a food composition for preventing or improving an inflammatory disease comprising (essentially) the strain or its culture as an active ingredient.
  • Another object of the present invention is to provide a cosmetic composition for preventing or improving inflammatory diseases comprising the strain or a culture thereof as an active ingredient.
  • compositions for preventing or improving an inflammatory disease comprising (essentially) the strain or its culture as an active ingredient.
  • Another object of the present invention is to provide a dressing composition comprising the strain or a culture thereof as an active ingredient.
  • a dressing composition comprising (essentially) the strain or a culture thereof as an active ingredient.
  • Another object of the present invention is to provide a probiotic composition comprising the strain or a culture thereof as an active ingredient.
  • probiotic composition consisting of the strain or a culture thereof as an active ingredient.
  • Another object of the present invention is to provide a composition for immunomodulation comprising the strain or a culture thereof as an active ingredient.
  • composition for immunomodulation consisting of the strain or a culture thereof as an active ingredient.
  • Another object of the present invention is to provide a food additive composition comprising the strain or a culture thereof as an active ingredient.
  • composition for food additives (essentially) composed of the above-described strain or a culture thereof.
  • Another object of the present invention is to provide the use of the strain or its culture for preparing an agent for the prevention or treatment of inflammatory diseases.
  • Another object of the present invention is to provide a method of treating an inflammatory disease, comprising administering an effective amount of a composition comprising the strain or a culture thereof as an active ingredient to an individual in need thereof.
  • Another object of the present invention is to provide a use of the strain or its culture for producing a probiotic.
  • Another object of the present invention is to provide the use of the strain or its culture for preparing a preparation for dressing.
  • Another object of the present invention is to provide the use of the strain or its culture for preparing an agent for immunomodulation.
  • Another object of the present invention is to provide a method of immunomodulation comprising administering an effective amount of a composition comprising the strain or a culture thereof as an active ingredient to an individual in need thereof.
  • Another object of the present invention is to provide a use of the strain or a culture thereof for preparing a food additive preparation.
  • the present invention provides a strain of Kazachstania turicensis CAU Y1706 (Accession No.: KCTC13794BP).
  • the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising the strain or a culture thereof as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising (essentially) the strain or a culture thereof as an active ingredient.
  • the present invention provides a food composition for preventing or improving inflammatory diseases comprising the strain or a culture thereof as an active ingredient.
  • the present invention provides a food composition for preventing or improving inflammatory diseases comprising (essentially) the strain or its culture as an active ingredient.
  • the present invention provides a cosmetic composition for preventing or improving inflammatory diseases comprising the strain or a culture thereof as an active ingredient.
  • the present invention provides a cosmetic composition for preventing or improving inflammatory diseases comprising (essentially) the strain or its culture as an active ingredient.
  • the present invention provides a dressing composition comprising the strain or a culture thereof as an active ingredient.
  • the present invention provides a dressing composition comprising (essentially) the strain or a culture thereof as an active ingredient.
  • the present invention provides a probiotic composition comprising the strain or a culture thereof as an active ingredient.
  • the present invention provides a probiotic composition (essentially) comprising the strain or a culture thereof as an active ingredient.
  • the present invention provides a composition for immunomodulation comprising the strain or a culture thereof as an active ingredient.
  • the present invention provides a composition for immunomodulation comprising (essentially) the strain or a culture thereof as an active ingredient.
  • the present invention provides a food additive composition comprising the strain or its culture as an active ingredient.
  • the present invention provides a composition for food additives (essentially) comprising the strain or a culture thereof as an active ingredient.
  • the present invention provides the use of the strain or its culture for preparing a preparation for the prevention or treatment of inflammatory diseases.
  • the present invention provides a method of treating an inflammatory disease comprising administering an effective amount of a composition comprising the strain or a culture thereof as an active ingredient to an individual in need thereof.
  • the present invention provides the use of the strain of claim 1 or a culture thereof for producing a probiotic.
  • the present invention provides the use of the strain or a culture thereof for preparing a preparation for dressing.
  • the present invention provides the use of the strain or its culture for preparing an agent for immunomodulation.
  • the present invention provides a method of immunomodulation comprising administering an effective amount of a composition comprising the strain or a culture thereof as an active ingredient to an individual in need thereof.
  • the present invention provides the use of the strain or its culture for preparing a food additive preparation.
  • the present invention kann Turismensis ( kazachstania turicensis ) CAU Y1706 (Accession number: KCTC13794BP ) Strain.
  • the Kazaztania thuricensis is a strain characterized by having anti-inflammatory activity.
  • the imbalance of the Th1/Th2 immune response in particular the Th2 immune response It is characterized by improving the imbalance of the excess immune response.
  • the present inventors isolate the strain of the present invention from kimchi, significantly reduce the concentration of Th2 cytokines such as IL-4 and IL5, and the levels of Th1 cytokines such as IFN- ⁇ , TNF- ⁇ , IL-12 are A novel yeast Kazaztania thuricensis CAU Y1706 that inhibits Th2 cytokines was selected by increasing and also reducing Treg cytokine levels.
  • the selected strain was analyzed by molecular biology using 18s, 26s rRNA sequencing, and confirmed to be a novel Kazaztania thuricensis yeast, and named as CAU Y1706. This strain was deposited on January 22, 2019 at the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center (KCTC) (Accession No.: KCTC13794BP).
  • Kazachstania turicensis according to the present invention is conventionally called saccharomyces turicensi s, and is known as yeast present in kefir grains of healthy fermented milk.
  • the novel yeast of the present invention not only regulates immunocytokines, but also reduces IgE, eosinophils, neutrophils, basophils in serum, and also regulates microbial flora in the intestine.
  • the novel yeast of the present invention showed an effect of improving inflammation in skin and intestine in a model using ovalbumin-sensitized mice. Specifically, it was confirmed that it suppresses the accumulation of mast cells in back skin cells and ileal lesions and also reduces eosinophil counts. In addition, it was confirmed that the eosinophil infiltration level of the back skin was also reduced.
  • novel yeast according to the present invention is also excellent in acid resistance and bile resistance.
  • the new yeast according to the present invention is not only excellent in anti-inflammatory effect, particularly in reducing skin inflammation, but also has a suit effect, and has excellent acid resistance and bile resistance, so it can reach the intestine excellently, so its commercial use value is high. .
  • the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising the strain or a culture thereof as an active ingredient .
  • the term “comprising” is used in the same sense as “including” or “characterized by,” and refers specifically to the composition or method according to the present invention. Do not exclude additional components or method steps that have not been made. In addition, the term “consisting of” means excluding additional elements, steps or components, which are not described separately.
  • the term “essentially consisting of” means that in the scope of a composition or method, it may include substances or steps that do not materially affect the basic properties thereof, in addition to the materials or steps described.
  • the'kazachstania turicensis CAU Y1706' or the'kazachstania turicensis CAU Y1706 cell' is not only the live cells that have been separated or purified from the culture medium, but also , which includes any processing form of lactic acid bacteria known to those skilled in the art (especially processing form of live cells), and includes, for example, dried materials, frozen materials, and the like. In addition, it may be used in a culture medium mixed with one or more compounds that are easily compounded with the strain, or in a non-mixed form.
  • the strain according to the present invention may be used in a living form, a semi-active form, an inactivated form, or a killed form, but is preferably a living form.
  • a living form it can be selected and used without limitation as long as it is by a method for lactic acid bacteria fertilization known in the art.
  • 'Cultivation' in the present invention means all actions performed in order to grow microorganisms in an artificially controlled environment, and in the present invention, the concept includes'fermentation', and'culture' is'fermentation'. It includes a mean, and includes a culture medium cultured in the medium itself (that is, the culture medium itself containing bacteria), or a product derived from the culture medium itself, such as a result of processing such as heat treatment to the culture medium.
  • the inflammatory disease in the present invention is a Th1-mediated immune disease or Th2-mediated immune disease, more specifically, acute or chronic organ transplant rejection, graft versus host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, inflammatory skin disease, Multiple sclerosis, pancreatitis, traumatic shock, bronchial asthma, allergic rhinitis, allergic conjunctivitis, cystic fibrosis, acute bronchitis, chronic bronchitis, acute bronchiolitis, chronic bronchiolitis, osteoarthritis, gout, spondylosis, ankylosing spondylitis, Reiter syndrome, Psoriatic arthrosis, bowel disease spondylitis, combustive arthrosis, combustive ankylosing spondylitis, reactive arthritis, infectious arthritis, post-infectious arthritis, Lou Gehrig's disease, nodular polyarteritis, irritable vasculitis, Lou Gehrig's granul
  • transplant rejection refers to a patient transplanted after transplantation of a solid organ such as a heart, lung, heart and lung complex, liver, kidney, pancreas, skin, bowel, or cornea.
  • Acute or chronic transplant rejection caused by cell death and tissue necrosis by invading and attacking immune cells in the body, and graft-versus-host disease (GVHD) after bone marrow transplantation Can.
  • GVHD graft-versus-host disease
  • inflammatory skin diseases include psoriasis, atopic dermatitis, eczematous dermatitis, contact dermatitis, seborrheic dermatitis, rosaceous nasal stiffness, lichen planus, vasculitis, pore red nasal hypertension, cellulitis, folliculitis, carcinoma, pemphigus, bullous ) It is characterized by one or more diseases selected from the group consisting of celestial swelling, epidermal vesicles, dammajin, angioedema, vasculitis, erythema and skin eosinophilia.
  • prevention in the present invention means all actions to suppress or delay the development of degenerative brain disease by administration of the pharmaceutical composition according to the present invention, and “treatment” means degenerative brain disease by administration of the pharmaceutical composition. It means any action that the symptoms of the suspected and affected individual are improved or beneficially altered.
  • the content of the composition is not greatly limited depending on the purpose or aspect of use, for example, 0.01 to 99% by weight, preferably 0.5 to 50% by weight, more preferably 1 to 30 based on the total weight of the composition. Weight percent.
  • the pharmaceutical composition according to the present invention may further include an additive such as a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient.
  • the pharmaceutical composition according to the present invention may contain a pharmaceutically effective amount of the strain alone or may further include one or more pharmaceutically acceptable carriers.
  • the pharmaceutical composition of the present invention may be administered to a patient in a single dose, and may be administered by a fractionated treatment protocol in which multiple doses are administered for a long time.
  • the pharmaceutically effective amount in the above refers to an amount that exhibits a higher response than the negative control group, and preferably refers to an amount sufficient to treat or prevent degenerative brain disease.
  • the effective amount of the strain according to the present invention may be 0.001 to 1000 mg/kg b.w./day, preferably 1 to 2000 mg/kg b.w./day, but is not limited thereto. However, the pharmaceutically effective amount may be appropriately changed according to various factors such as the disease and its severity, the patient's age, weight, health status, sex, administration route, and treatment period.
  • composition of the present invention can be variously formulated according to the route of administration by a method known in the art together with the pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention may be administered orally or parenterally to mammals, including humans, according to the desired method, and the parenteral administration methods include external application to the skin, intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, and muscle. Intra-injection or intra-thoracic injection.
  • composition of the present invention can be variously formulated according to the route of administration by a method known in the art together with the pharmaceutically acceptable carrier.
  • the route of administration is not limited to this, but may be administered orally or parenterally, preferably oral administration.
  • the pharmaceutical composition of the present invention is a powder, granules, tablets, pills, dragees, capsules, liquids, gels according to methods known in the art together with a suitable carrier for oral administration. , Syrups, suspensions, wafers, and the like.
  • oral preparations include tablets, pills, powders, granules, capsules, and the like, and the active ingredient is combined with a solid excipient, then crushed and added with a suitable adjuvant, and then processed into a granule mixture to tablet or dragee tablets. Can be obtained.
  • excipients examples include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol, and starches including corn starch, wheat starch, rice starch, potato starch, and the like. Fillers such as cellulose, gelatin, polyvinylpyrrolidone, etc. may be included, including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose. In addition, if necessary, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may further include an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and a preservative.
  • sugars including lactose, dextrose, sucrose, sorbitol, mannitol, x
  • the pharmaceutical composition of the present invention when administered parenterally, may be formulated according to methods known in the art together with a suitable parenteral carrier.
  • Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories.
  • non-aqueous solvent and the suspension solvent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used.
  • injectable ester such as ethyl oleate
  • suppositories As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.
  • composition of the present invention can be administered in parallel with a known compound having an effect of preventing and treating degenerative brain disease.
  • the present invention provides a cosmetic composition for preventing or improving inflammatory diseases comprising the strain or a culture thereof as an active ingredient.
  • the cosmetic composition according to the present invention may be prepared in any formulation conventionally prepared in the art, and dermatology together with a dermatologically acceptable medium, base, adjuvant, excipient, etc. in addition to the novel strain or culture thereof according to the present invention
  • Basic cosmetic compositions commonly produced in the field face washes, creams, essences, cleansing agents such as cleansing foams and cleansing water, packs, body oils, mists, soaps
  • color cosmetic compositions foundation, lipstick, mascara, makeup base
  • hair Product composition shampoo, conditioner, hair conditioner, hair gel
  • soap may be prepared.
  • excipients are not limited to, for example, skin emollients, skin penetration enhancers, colorants, fragrances, emulsifiers, emollients, thickeners, thickeners, gelling agents, suspending agents, stabilizers, foaming agents, interfaces Active agents, fillers, or solvents.
  • fragrances, pigments, fungicides, antioxidants, preservatives, auxiliary agents and moisturizers may be further included, and for the purpose of improving physical properties, may include thickeners, inorganic salts, synthetic polymer materials, and the like.
  • the cleanser and soap with the cosmetic composition of the present invention can be easily prepared by adding the extract of the present invention to a conventional cleanser and soap base.
  • a cream it can be prepared by adding the strain of the present invention or a culture thereof to a cream base of a general oil-in-water type (O/W).
  • Synthetic or natural materials such as proteins, minerals, vitamins, etc. for the purpose of improving physical properties may be additionally added to flavors, chelating agents, pigments, antioxidants, preservatives, and the like.
  • Suitable cosmetic composition formulations include, for example, solutions, gels, solid or dough anhydrous products, emulsions obtained by dispersing an oil phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes), nonionic It may be provided in the form of a vesicle dispersant, cream, skin, lotion, powder, ointment, spray or conceal stick. It can also be prepared in the form of a foam or aerosol composition further containing a compressed propellant.
  • the product to which the cosmetic composition of the present invention can be added is not limited to, but is not limited to, skin lotion, skin softener, skin toner, converging makeup, softening makeup, nutritional makeup, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, body Cream, massage cream, nutrition cream, moisturizing cream, hand cream, essence, nutrition essence, pack, soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, treatment, essence, emulsion, press powder, Formulations such as rust powder, eye shadow, and the like.
  • the present invention provides a food composition for preventing or improving an inflammatory disease comprising the strain or a culture thereof as an active ingredient.
  • the food composition according to the present invention includes all forms of functional food, nutritional supplement, health food, health functional food and food additives. These types can be prepared in various forms according to conventional methods known in the art.
  • the food composition itself of the present invention may be prepared in the form of tea, juice, and drink to be consumed or granulated, encapsulated, and powdered.
  • the food composition of the present invention can be prepared in the form of a composition by mixing with a known substance or active ingredient known to have an effect of protecting skin cells or improving wrinkles.
  • the health food for example, can be processed into leached tea, liquid tea, beverage, fermented milk, cheese, jelly, yogurt, juice, probiotic preparations, and health supplements, and can be used in the form of various food additives according to other purposes Can.
  • the formulation of the dietary supplement may be in various forms, such as solutions, emulsions, viscous mixtures, tablets, powders, pills, etc., which may be administered by various methods such as simple drinking, injection, spray or squeeze. .
  • the food composition of the present invention has the advantage of having a more excellent effect by ingesting in the form of an inner beauty food.
  • the inner beauty is a food called'eating cosmetics or beauty food', refers to food that absorbs various ingredients that are good for the skin into the body and changes the skin's constitution to be healthy, and chooses cosmetics that fit the skin type. You can choose the inner beauty food that suits each individual and take it in consideration of the skin condition and lifestyle. More preferably, when the cosmetic composition containing the cosmetic composition and the inner beauty food containing the strain are mixed, the effect is significantly higher than that of using only the cosmetic product, which has the advantage of showing a more effective improvement effect of inflammatory diseases. .
  • functional foods include beverages (including alcoholic beverages), fruits and processed foods (e.g., canned fruits, canned foods, jams, marmalades, etc.), fish, meat, and processed foods (e.g. ham, sausage corn beef, etc.) ), breads and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), juice, various drinks, cookies, syrup, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, Retort food, frozen food, various seasonings (eg, miso, soy sauce, sauce, etc.) can be prepared by adding the food composition of the present invention.
  • the food composition of the present invention in the form of a food additive, it may be prepared and used in powder or concentrate form.
  • the strain content of the present invention which is an active ingredient in the food composition according to the present invention, is 0.01 to 99% by weight, preferably 0.1 to 50% by weight, more preferably 0.5 to 25% by weight, based on the total weight of the composition. It is not limited.
  • strain of the present invention in the form of a food additive, it may be prepared and used in the form of a powder or a concentrate.
  • the mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or symptom relief), but is not limited thereto, for example, the strain according to the present invention 10 8 CFU/g to 10 20 CFU/g.
  • the effective dose of the strain of the present invention may be used in accordance with the above concentration range, but in the case of long-term intake for health and hygiene purposes or for health control purposes, it may be less than the above range, and the active ingredient is safe Since there is no problem, it may be used in an amount exceeding the above range.
  • the present invention provides a probiotic or dressing composition comprising the strain or a culture thereof as an active ingredient.
  • the novel strain according to the present invention may be used as a probiotic for probiotic formulations for the promotion of human and animal health or a probiotic composition for feed for animals.
  • the composition includes the strain itself, or a culture thereof as an active ingredient, and may further include an excipient or a carrier.
  • the content of the novel strain of the present invention in the composition may vary depending on the composition and use of the composition.
  • compositions according to the present invention can be prepared and administered in a variety of formulations and methods.
  • a novel strain or a culture thereof according to the present invention is mixed with a carrier and a fragrance commonly used in the pharmaceutical field, and then tablets, troches, capsules, elixirs ), syrup, powder, suspension, or granules.
  • a carrier a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, etc. can be used.
  • the administration method may be oral, parenteral or application.
  • the dosage can be appropriately selected according to the absorbance of the active ingredient in the body, the rate of inactivation and excretion, the age, sex, and condition of the recipient.
  • novel strains or cultures thereof according to the present invention can be used as food additives for foods such as kimchi, beverages, baby foods, and in particular, as starters for manufacturing fermented products.
  • The'fermented product' may be referred to herein interchangeably with the term'fermented food'.
  • the fermentation product is a food that requires a fermentation process at the time of manufacture, and if known in the art, its type is not limited, for example, cheese, yogurt, butter, cream, ice cream, lactic acid bacteria drink, capeer, fermented milk, kimchi, fermentation Products and the like. Fermentation products using the novel strain of the present invention can be prepared according to conventional methods known in the art.
  • the method of manufacturing fermented products usually consists of the preparation of raw materials, the addition of lactic acid bacteria, fermentation, and recovery of the finished product.
  • the preparation step of the raw material is a step of preparing the material to be fermented and preparing the fermentation conditions so that the fermentation is performed well.
  • the addition of lactic acid bacteria is to add an appropriate amount of bacteria according to the amount of the fermentation target. Fermentation is performed according to the fermentation conditions of a conventional fermentation bacterium, and may be performed, for example, at 20°C to 40°C for 1 to 168 hours.
  • Recovery of the finished product includes removing unnecessary by-products or unfermented materials from the fermentation product and all post-processing procedures and packaging to facilitate storage and transportation.
  • the present invention provides a composition for immunomodulation comprising the strain or a culture thereof as an active ingredient.
  • Th1 cells producing interleukin-2 (IL-2), interferon ⁇ (IFN- ⁇ ), and TNF- ⁇ , and IL-4, IL-5, IL-6, IL -10, and Th-13 cells that produce IL-13.
  • IL-2 interleukin-2
  • IFN- ⁇ interferon ⁇
  • Th13 Th13 cells that produce IL-13.
  • Immune balance regulated by cytokines produced by these Thl and Th2 cells is called Thl/Th2 balance
  • Thl cells are important for the regulation of cellular immunity
  • Th2 cells are known to play an important role in the regulation of humoral immunity. have.
  • the cytokine centering on IFN- ⁇ which is important for the differentiation of Thl
  • the cytokine mainly IL-4 which is important for the differentiation of Th2
  • the Thl/Th2 balance is broken, it can cause various immune diseases.
  • cellular immunity is revived to increase infection resistance, and when to Th2 bias, infection resistance decreases, and conversely, allergy increases. do.
  • the yeast according to the present invention inhibits Th2 cytokines (eg IL-4, IL-5) or/and induces Th1 cytokines (eg IL-12, IFN- ⁇ , TNF- ⁇ ) Th1/ It has a physiological activity that regulates the balance of the Th2 immune response.
  • the specific physiological activity is as follows. 1) increase production of IL-12, IFN- ⁇ , TNF- ⁇ in ovalbumin-sensitized mice; 2) Reduce IgE levels in mouse serum. 3) Reduce the ratio of eosinophils, neutrophils, and basophils. 4) inhibit the expression of cytokines (IL-4 and IL-10) mediated by Th2-lymphocytes; 5) Reduces eosinophil and mast cell infiltration at the site of atopic dermatitis lesions.
  • the present invention provides a food additive composition comprising the strain or a culture thereof as an active ingredient.
  • the strain of the present invention or cultures thereof according to the present invention can be used as a food additive for foods such as kimchi, beverages, baby food, fermented milk, and bread.
  • the strain or culture of the present invention may be used as a starter for manufacturing fermented products.
  • the fermented products include cheese, glyphchi, fermented products, and the like.
  • the fermentation product using the strain of the present invention or a culture thereof may be prepared according to a conventional method known in the art.
  • fermented reproductive products can be prepared by appropriately blending various agricultural products such as baektae, glutinous rice, and sorghum to have excellent nutritional balance and palatability.
  • the present invention provides the use of the strain or a culture thereof for preparing an agent for the prevention or treatment of inflammatory diseases.
  • the present invention provides a method of treating an inflammatory disease comprising administering an effective amount of a composition comprising the strain or culture thereof as an active ingredient to an individual in need thereof.
  • the present invention provides the use of the strain or its culture for producing a probiotic.
  • the present invention provides the use of the strain or its culture for preparing a formulation for dressing.
  • the present invention provides the use of the strain or its culture for preparing an agent for immunomodulation.
  • the present invention provides a method of immunomodulation comprising administering an effective amount of a composition comprising the strain or culture thereof as an active ingredient to an individual in need thereof.
  • the present invention provides the use of the strain or its culture for preparing a food additive preparation.
  • The'effective amount' of the present invention refers to an amount that, when administered to an individual, shows an effect of improving, treating, preventing, detecting, diagnosing or suppressing or reducing an inflammatory disease
  • the'individual' is an animal, preferably It may be an animal including mammals, particularly humans, or cells, tissues, organs, etc. derived from animals.
  • the subject may be a patient in need of the effect.
  • The'treatment' of the present invention broadly refers to improving the symptoms of an inflammatory disease or inflammatory disease, which may include curing, substantially preventing, or improving the condition, from the disease It includes, but is not limited to, alleviating, healing or preventing one symptom or most of the symptoms.
  • the novel yeast according to the present invention promotes a Th2 immune response mediated by Treg, has anti-inflammatory activity, has a formal effect, and has an excellent effect of suppressing inflammation in the skin and intestine against the ovalbumin-sensitized mouse model. In addition, it has excellent acid resistance and bile resistance, and thus has high commercial value.
  • FIG. 1 is a diagram showing a design and a schedule of atopic dermatitis induction experiment in OVA-sensitized mice for the experiment.
  • FIG. 2 is a scanning electron microscope (SEM) photograph of Kauriztania Turisensis CAU Y1706.
  • 3A to 3G are results confirming the effect of oral administration of Kazaztania Turisensis CAU Y1706 on cytokine concentration. Serum was used to analyze the values, and an ELIZA kit was used. Signs of significance indicate the difference between the ANOVA analysis value of the positive group and the mean value. *p ⁇ 0.05; **p ⁇ 0.005; ***p ⁇ 0.0005; ****p ⁇ 0.0001.
  • Figure 3a is a result confirming the effect on the IL-4 concentration.
  • Figure 3b is a result confirming the effect on the IL-5 concentration.
  • Figure 3c is a result confirming the effect on the IL-10 concentration.
  • Figure 3d is a result confirming the effect on the IL-12 concentration.
  • Figure 3e is a result confirming the effect on the IFN- ⁇ concentration.
  • Figure 3f is a result confirming the effect on the TNF- ⁇ concentration.
  • Figure 3g is a result confirming the effect on the concentration of IL-1 ⁇ .
  • 4A to 4E are results confirming the effect of oral administration of Kazaztania Turisensis CAU Y1706 on blood. Inflammation was confirmed by whole blood analysis. Signs of significance indicate the difference between the ANOVA analysis value of the positive group and the mean value. * p ⁇ 0.05; ** p ⁇ 0.005; *** p ⁇ 0.0005; **** p ⁇ 0.0001.
  • Figure 4a is a result of measuring the IgE level in the serum by ELISA analysis to confirm the allergic reaction.
  • Figure 4b is the result confirming the eosinophil ratio.
  • Figure 4e is a result of checking the basophil ratio.
  • 5A to 5H are results confirming that oral administration of Kazaztania thuricensis CAU Y1706 contributes to the abundance and diversity of the intestinal microbial population of atopic dermatitis-induced mice.
  • Figure 5a is a result of measuring the Shannon-wiener index (defined by 97% similarity).
  • 5B is a result of measuring the Simpson index.
  • Figure 5c is a result of estimating the difference in the intestinal microflora in an Unweighted UniFrac based (PCoA) 3D chart (blue label: negative control; red label: positive control; green label: K. turicensis CAU Y1706 administered group; purple label: betota Stinbesylate group).
  • PCoA Unweighted UniFrac based
  • Figure 5d is a result showing the relative abundance of the genera unit.
  • Figure 5e is a result showing the scatter plot of Faecalibacterium genus.
  • Figure 5f is a result showing the scatter plot of the genus Akkermansia.
  • Figure 5g is a result showing the scatter plot in the genus Lactobacillus.
  • Figure 5h is a result showing the scatter plot in the genus Ruminococcus.
  • 6A to 6D are results confirming the effect of oral administration of Kazaztania Turisensis CAU Y1706 on atopic dermatitis skin lesions in rats. Signs of significance indicate the difference between the ANOVA analysis and the mean value of the positive group. *p ⁇ 0.05; **p ⁇ 0.005; ***p ⁇ 0.0005; ****p ⁇ 0.0001.
  • FIG. 6A shows a dermatitis index for atopic lesions of skin, such as mice, and dermatitis scores are calculated as the sum of the scores of the three symptoms of erythema, dryness, and scratching of mice.
  • Figure 6d is a result of quantifying the number of eosinophils through microscopic analysis after staining skin tissues such as mice with Congo red.
  • Kimchi samples were obtained from a traditional market in Seoul, Korea. All samples were chopped and mixed with 10 mL of peptone solution (0.85% mass/vol). After the stepwise dilution, the samples are spread on MRS (Man, Rogosa, and Sharpe; BD BBL, Spatks, MD. USA) solid medium and 2-3 in a MIR-254-PK incubator at 30°C (Panasonic, Osaka, Japan). Cultured daily. For pure separation, white colonies were screened and subjected to several separation cultures on Tryptic Soy Agar-TSA (BD BBL), and all separation cultures were performed at 30° C. for 24 hours.
  • MRS Man, Rogosa, and Sharpe; BD BBL, Spatks, MD. USA
  • MIR-254-PK incubator at 30°C (Panasonic, Osaka, Japan). Cultured daily. For pure separation, white colonies were screened and subjected to several separation cultures on Tryptic Soy Agar-TSA (BD BBL),
  • rRNA gene (18S, 26S, 5.8-ITS) sequence analysis was used, and 3730 automatic DNA sequencer (Applied Biosystems, Foster City, CA, USA) was used for analysis. Gene sequences were analyzed via NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The screened yeast was cultured in TSA at a condition of 30° C., and then used an optical microscope DM 1000 light (Leica, Wetzlar, Germany) and a scanning electron microscope SIGMA field-emission scanning electron microscope (Carl Zeiss, Dresden, Germany). Cell appearance was observed.
  • the thus-selected Kazaztania thuricensis strain was named CAU Y1706.
  • the newly identified strains of Kazaztania thuricensis were deposited with the Korea Research Institute of Bioscience and Biotechnology Biotechnology Center (KCTC) on January 22, 2019 (Accession No.: KCTC13794BP).
  • mice Animal experiments were conducted in accordance with the guidelines of the Korea Food and Drug Safety Management Headquarters. Rat blood samples and tissue samples were collected according to the guidelines of Chung-Ang University Animal Bioethics Committee. Female BALB/c rats (40 animals), 5 weeks old, were pre-sold in central laboratory animals (Seoul, Korea). The experimental design and planning table are shown in FIG. 1 . All mice were negative control (negative control, no OVA + PBS), positive control (positivw control, OVA sensitization + PBS), K. turicensis CAU Y1706 group (OVA sensitization + K.
  • bepotastine besylate group (OVA sensitization + bepotastine besylate) were randomly assigned to each of 10 animals in four groups.
  • OVA sensitization + bepotastine besylate were randomly assigned to each of 10 animals in four groups.
  • OVA sensitization + bepotastine besylate were randomly assigned to each of 10 animals in four groups.
  • OVA sensitization + bepotastine besylate were randomly assigned to each of 10 animals in four groups.
  • OVA sensitization + bepotastine besylate were randomly assigned to each of 10 animals in four groups.
  • OVA sensitization + bepotastine besylate were randomly assigned to each of 10 animals in four groups.
  • OVA sensitization + bepotastine besylate were randomly assigned to each of 10 animals in four groups.
  • OVA sensitization + bepotastine besylate were randomly assigned to each of 10 animals in four groups.
  • the skin tissue and ileum of the victim's back were used for tissue analysis.
  • the tissue was fixed in a formalin solution diluted to 10% with phosphate buffered saline, and then embedded in paraffin.
  • Tissue samples were cut using a microtome to a thickness of 4-5 ⁇ m, and stained with hematoxylin and eosin, toluidine blue and congo red for mast cells and eosinophil measurements, respectively. All toluidine blue staining sites of the ileum tissue samples for mast cell count were observed at 400x magnification by DM 4000B microscopy.
  • 5 of the toluidine blue staining regions of the skin tissue of the back were randomly selected, and 20 of the congo red staining regions were randomly selected for quantification of eosinophils.
  • GraphPad Prism (v.7.0) software was used for statistical analysis, and data were expressed as mean ⁇ standard error. Comparisons between groups to identify the relative differences between microbial rivers and genus were based on statistically significant differences by the nonparametric Kruskal-Wallis test. Significant differences in cytokine, blood sample, and tissue analysis were calculated by ANOVA analysis to compare two or more groups, and were considered to be significant when the p value was less than 0.05.
  • K. turicensis CAU Y1706 formed a round, shiny creamy colony when cultured in TSA medium (Bacto) for one day in an environment of 30°C. The cells were oval and the diameter was observed to be 1.6-2.3 ⁇ m and the length was 1.6-2.3 ⁇ m (FIG. 2 ). K. turicensis CAU Y1706 was confirmed to be able to utilize D-glucose, D-galactose, D-saccharose, D-trehalose, D-raffinose, D-fructose, D-mannose, 2-keto-D-gluconate as a carbon source. Became.
  • K. turicensis CAU Y1706 The effect of K. turicensis CAU Y1706 on serum cytokine production was examined. Compared to the positive control group, the concentrations of Th2 cytokines such as IL-4 and IL-5 in the serum of the K. turicensis CAU Y1706 administration group were significantly lower. The group administered with bepotastine besylate also showed low levels (FIGS. 3A and 3B ). Compared to the positive control group, Treg cytokine levels such as IL-10 and IL-1 ⁇ in the serum were also lower in the K. turicensis CAU Y1706 administration group (FIGS. 3C and 3G ).
  • the group treated with the strain of the present invention has superior IFN- ⁇ , TNF- ⁇ , IL-12 production compared to the control group.
  • Serum IgE levels were higher in the positive control group and K. turicensis In the CAU Y1706-administered group and the bepotastine besilate-administered group, there was a noticeably lowered value (FIG. 4A).
  • the positive control, bepotastine besylate group and K. turicensis When comparing the CAU Y1706 administration group, K. turicensis It was confirmed that the ratio of eosinophils, eosinophils, neutrophils, and basophils in the CAU Y1706 administration group was remarkably decreased (FIGS. 4B-4E ).
  • bacterial DNA was extracted from the fecal sample, and the 16S rRNA gene sequence was amplified through PCR. On average, an average of 599 bp ( ⁇ 9.2 bp) of bacterial sequence was 80.778 per sample, and a total of 3,231,137 were obtained.
  • the diversity and abundance of bacterial populations were statistically analyzed through operational taxonomic units (OTUs) (defined as 97%) and Shannon and Simpson indices (FIGS. 5A and 5B ).
  • OTUs operational taxonomic units
  • FIGS. 5A and 5B Shannon and Simpson indices
  • Faecalibacterium contained in Firmicutes was found to be lower in the negative and K. turicensis CAU Y1706 groups compared to the positive control group (p ⁇ 0.0001). Conversely, Ruminococcus showed higher values in the negative and K. turicensis CAU Y1706 groups compared to the positive control group (p ⁇ 0.05).
  • SCFAs short chain fatty acids
  • SCFA butyrat plays an important role in innate immunity and has functions such as protecting intestinal health, anti-inflammatory effects, and promoting Treg cell differentiation.
  • Bacteroides , Ruminococcus , and Akkermansia are known to produce butyrat
  • Lactobacillus is known to be involved in promoting butyrat production.
  • Example 5 skin inflammation and intestines Kazachstania turicensis CAU Impact of Y1706
  • the Kazachstania After inoculating the turicensis CAU Y1706 strain into TSB liquid medium, incubating for 12 hours at 30°, and recovering the cells by centrifugation, adjusting the OD 600 value to 1.0 with sterile water to adjust the suspension 30 ⁇ l to pH 2.0 TSB liquid After inoculating the medium and incubating at 30°C for 0-120 minutes, the viable cell count was measured at A600 nm with Nanodrop (NanoQuant infinite M200, TECAN).
  • CAU Y1706 showed the ability to grow in MRS medium at pH 2.0 (same condition as gastric juice). CAU Y1706 confirmed that over time, the number of bacteria increased at a significant rate, and thus it was not inhibited under strong acid conditions. Therefore, it was confirmed that CAU Y1706 of the present invention has excellent acid resistance.
  • Kazachstania In order to test the resistance characteristics of turicensis CAU Y1706 to bile acids, strains were inoculated into TSB liquid medium containing 0, 0.2 and 0.4% (w/v) bile acids (ox-bile dry pure, Merck, Germany) and cultured. After that, the viable cell count was measured. More specifically, the Kazachstania After inoculating the turicensis CAU Y1706 strain into the MRS liquid medium, incubating at 30° C.
  • the viable cell count was measured at A600 nm with Nanodrop (NanoQuant infinite M200, TECAN).
  • Kazachstania turicensis CAU Y1706 showed the ability to grow in TSB medium to which 0.2% or 0.4% of bile was added, and the bacteria showed excellent growth ability in 0.2% and 0.4% of bile.
  • CAU Y1706 was found to have a high bile resistance (BTR) ability of 1.25 (0.2% bile condition) and 1.05 (0.4% bile condition) compared to the control group without addition of bile. Therefore, it was confirmed through the test that the strain of the present invention stably passes through the digestive system and a large number of bacteria can survive and reach the intestine.
  • BTR bile resistance
  • the novel yeast according to the present invention promotes a Th2 immune response mediated by Treg, has anti-inflammatory activity, has a formal effect, and has an excellent effect of suppressing inflammation in the skin and intestine against the ovalbumin-sensitized mouse model. In addition, it has excellent acid resistance and bile resistance, and thus has high industrial applicability.

Abstract

The present invention relates to a novel Kazachstania turicensis CAU Y1706 and a composition using same, and more specifically, to a Kazachstania turicensis CAU Y1706 (Accession No.: KCTC13794BP) strain, and a pharmaceutical composition, a food composition, a cosmetic composition, and a probiotic composition for preventing or treating inflammatory diseases, comprising the strain or a culture thereof as an active ingredient.

Description

신규한 카자츠타니아 투리센시스 CAU Y1706 및 이를 이용한 조성물Novel Kazaztania Turisensis CAU Y1706 and composition using same
본 출원은 2019년 1월 23일에 출원된 대한민국 특허출원 제10-2019-0008823호를 우선권으로 주장하고, 상기 명세서 전체는 본 출원의 참고문헌이다.This application claims priority to Korean Patent Application No. 10-2019-0008823 filed on January 23, 2019, and the entire specification is a reference to this application.
본 발명은 신규한 카자츠타니아 투리센시스 CAU Y1706 및 이를 이용한 조성물에 관한 것으로, 보다 상세하게는 카자츠타니아 투리센시스 CAU Y1706 (수탁번호 : KCTC13794BP) 균주, 그리고 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 약학적 조성물, 식품 조성물, 화장료 조성물, 그리고 생균제 조성물에 대한 것이다. The present invention relates to a novel Kazaztania thuricensis CAU Y1706 and a composition using the same, and more specifically, the Kazaztania thuricensis CAU Y1706 (Accession No.: KCTC13794BP) strain, and the strain or a culture thereof are effective. It relates to a pharmaceutical composition, a food composition, a cosmetic composition, and a probiotic composition for the prevention or treatment of inflammatory diseases including as a component.
염증(inflammation)은 몸을 구성하는 세포에 파괴를 주는 연속적이고 복합적인 반응이다. 염증 반응은 외부 감염원(박테리아, 곰팡이, 바이러스, 다양한 알레르기 유발물질)의 침입에 의해 형성되는 병리적 상태를 말하며, 이러한 생체의 세포나 조직에 어떠한 기질적 변화를 가져오는 침습이 가해질 때 그 손상부위를 수복 재생하려고 하는 생체의 방어 반응과정이다.Inflammation is a continuous and complex reaction that destroys cells in the body. Inflammatory reaction refers to a pathological condition formed by the invasion of external infectious agents (bacteria, fungi, viruses, and various allergens), and the damage site when an invasion that causes any organic changes to cells or tissues of these organisms is applied Is a defense reaction process of a living body that is trying to restore and regenerate.
일반적으로 염증반응은 생체의 세포나 조직에 어떠한 기질적 변화를 가져오는 침습으로 인한 손상을 재생하기 위한 생체방어 기작이고, 이 반응과정에서는 국소의 혈관, 체액의 각종 조직세포 및 면역세포 등이 작용한다. 정상적으로 외부 침입균에 의해 유도되는 염증반응은 생체를 보호하기 위한 방어 작용인 반면, 비정상적으로 과도한 염증반응이 유도되는 경우 다양한 질환들이 나타나게 된다.In general, an inflammatory reaction is a biological defense mechanism to regenerate damage due to invasion, which causes any organic changes in cells or tissues of a living body. In this reaction process, local blood vessels, various tissue cells of body fluids, and immune cells act do. While the inflammatory reaction normally induced by external invading bacteria is a protective action to protect the living body, various diseases appear when abnormally excessive inflammatory reaction is induced.
만성적 염증 질병 중 하나인 천식은 기도 과민반응(airway hyperresponsiveness), 폐 조직으로 염증세포의 침착, 점액의 과다 생산에 의해 일어나며, 인터루킨(IL)-4, IL-5, IL-13 등을 포함한 사이토카인과, 에오텍신, 렌티스 등의 케모카인이 과다 발현되면서 알레르기성 천식이 유도된다.Asthma, one of chronic inflammatory diseases, is caused by airway hyperresponsiveness, deposition of inflammatory cells into the lung tissue, and overproduction of mucus, and cytokines including interleukin (IL)-4, IL-5, and IL-13. Allergic asthma is induced by overexpression of chemokines such as cain, eotexin, and lentis.
활성이 된 면역세포인 B 세포에는 비만세포와, 호산성구(eosinophils) 세포가 다양한 염증 유도 매개체를 생산한다. 호산성구 세포는 천식발병의 기초적인 영향 매개체로써 미립의 단백질을 증가시키고, 활성산소종(reactive oxygen species)을 생성시킨다.In the activated immune cell, B cells, mast cells and eosinophils cells produce various inflammation-inducing mediators. Eosinophilic cells increase the amount of particulate proteins and create reactive oxygen species as the primary mediator of asthma development.
알레르기란 광범위하고 복잡한 병리적 현상의 총화로 면역반응에 근거한 생체의 전신적 또는 국소적인 장애이다. 인체에 나타나는 알레르기는 면역 기전에 따라 Ⅰ, Ⅱ, Ⅲ 및 Ⅳ형으로 분류되며, 이 중에서 즉시형 과민반응에 속하는 Ⅰ형 알레르기가 임상에 있어서 중요한 부분을 차지하고 있으며, 아토피성 피부염, 알레르기성 비염, 기관지 천식, 고초열 및 화분증 등이 여기에 속한다.Allergy is a systemic or local disorder of a living body based on an immune response that is the generalization of a wide range of complex pathological phenomena. Allergies in the human body are classified into Ⅰ, Ⅱ, Ⅲ, and Ⅳ types according to the immune mechanism, among which type I allergies belonging to the immediate hypersensitivity reaction occupy an important part in clinical practice, atopic dermatitis, allergic rhinitis, These include bronchial asthma, hyperthermia and pollinosis.
Ⅰ형 알레르기의 대표적 질환으로 알려진 천식(asthma)은 대부분 알레르기성 질환으로 기도의 광범위한 협착에 의해 발생하는 천명, 호흡곤란, 기침 등의 임상 증세들은 자연히 혹은 치료에 의해 가역적으로 호전될 수 있다. 천식으로 인한 사망자 수가 세계적으로 증가하고 있으며, 개발국에서는 3~10％가 천식을 앓고 있다.Asthma, which is known as a representative disease of type I allergies, is an allergic disease. Clinical symptoms such as wheezing, dyspnea, and cough caused by widespread narrowing of the airways can be reversibly improved naturally or by treatment. The number of deaths from asthma is increasing worldwide, and 3-10% of developing countries suffer from asthma.
또한, 알레르기 항원에 피부가 노출되면 랑게르한스 세포의 표면에 있는 IgE 수용체에 항원 특이 IgE가 결합하고, 이 항원의 표면에 있는 T세포에 전달되어 T세포가 활성화된다. 이 경우 정상과 달리 알레르기성 피부질환, 특히, 아토피 피부염에서는 Th2가 활성화되어 IL-4, IL-5, IL-6, IL-8, IL-10 및 IL-13 등의 사이토카인이 분비되고, 그 결과 B 세포에서 IgE의 생성이 촉진되고, 활성화되어있는 비만세포의 탈과립을 촉진시켜 염증성 사이토카인과 히스타민이 유리된다.In addition, when the skin is exposed to the allergen, antigen-specific IgE binds to the IgE receptor on the surface of Langerhans cells, and is delivered to T cells on the surface of this antigen to activate T cells. In this case, unlike normal, Th2 is activated in allergic skin diseases, especially atopic dermatitis, and cytokines such as IL-4, IL-5, IL-6, IL-8, IL-10 and IL-13 are secreted, As a result, the production of IgE in B cells is promoted, and degranulation of activated mast cells is promoted, inflammatory cytokines and histamine are released.
그러나, 지금까지 프로바이오틱스 효모가 염증성질환, 알러지, 특히 아토피 피부염에 미치는 영향에 대한 보고는 거의 없었다. However, there have been few reports of the effects of probiotic yeast on inflammatory diseases, allergies, and especially atopic dermatitis.
케피어는 유산균과 효모가 우유에서 공생발효하여 만들어지는 유제품으로, 잘 알려져 있는 프로바이오틱스 식품이다. 케피어 효모에 속해있는 카자츠타니아 투리센시스는 이전에는 사카로마이세스 투리센시스(saccharomyces turicensis)로 알려져 있었으며, 다양한 케피어 그레인으로부터 분리된다(System. Apple. Microbiol. 22, 420-425(1999).Kefir is a well-known probiotic food that is produced by symbiotic fermentation of lactic acid bacteria and yeast in milk. Kazaztania Turisensis, which belongs to kefir yeast, was previously known as saccharomyces turicensis and is isolated from various kefir grains (System. Apple.Microbiol. 22, 420-425 ( 1999).
그러나 한국의 전통음식인 김치에서 분리된 효모인 카자츠타니아 투리센시스 및 그의 염증성 질환에 대한 치료 효과 및 면역 조절능은 보고된 바 없었다.However, there has been no report on the therapeutic effect and immunomodulatory effects of yeast isolated from traditional Korean food kimchi, kazaztania thuricensis and its inflammatory diseases.
이에 본 발명자들은 면역반응을 조절하고, 오브알부민(OVA)-감작 AD 쥐 모델에서의 면역반응 조절 능력과 장내미생물군을 변화시키는 능력이 뛰어난 신규한 카자츠타니아 투리센시스(kazachstania turicensis) CAU Y1706를 분리하여 본 발명을 완성하였다.Accordingly, the present inventors regulate the immune response, and the novel ability to modulate the immune response in the ovalbumin (OVA)-sensitized AD rat model and the ability to change the intestinal microflora is a new kazachstania turicensis. The present invention was completed by separating CAU Y1706.
따라서 본 발명의 목적은 카자츠타니아 투리센시스(kazachstania turicensis) CAU Y1706(수탁번호 : KCTC13794BP) 균주를 제공하는 것이다.Accordingly, an object of the present invention is to provide a strain of Kazachstania turicensis CAU Y1706 (Accession No.: KCTC13794BP).
본 발명의 다른 목적은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases comprising the strain or a culture thereof as an active ingredient.
또한 상기 균주 또는 이의 배양물을 유효성분으로 (필수적으로) 이루어지는 염증성 질환의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.In addition, it is to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising (essentially) the strain or a culture thereof as an active ingredient.
본 발명의 다른 목적은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 염증성 질환의 예방 또는 개선용 식품 조성물을 제공하는 것이다. Another object of the present invention is to provide a food composition for preventing or improving inflammatory diseases comprising the strain or a culture thereof as an active ingredient.
또한 상기 균주 또는 이의 배양물을 유효성분으로 (필수적으로) 이루어지는 염증성 질환의 예방 또는 개선용 식품 조성물을 제공하는 것이다.In addition, it is to provide a food composition for preventing or improving an inflammatory disease comprising (essentially) the strain or its culture as an active ingredient.
본 발명의 또 다른 목적은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 염증성 질환의 예방 또는 개선용 화장료 조성물을 제공하는 것이다.Another object of the present invention is to provide a cosmetic composition for preventing or improving inflammatory diseases comprising the strain or a culture thereof as an active ingredient.
또한 상기 균주 또는 이의 배양물을 유효성분으로 (필수적으로) 이루어지는 염증성 질환의 예방 또는 개선용 화장료 조성물을 제공하는 것이다.In addition, it is to provide a cosmetic composition for preventing or improving an inflammatory disease comprising (essentially) the strain or its culture as an active ingredient.
본 발명의 또 다른 목적은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 정장용 조성물을 제공하는 것이다.Another object of the present invention is to provide a dressing composition comprising the strain or a culture thereof as an active ingredient.
또한 상기 균주 또는 이의 배양물을 유효성분으로 (필수적으로) 이루어지는 정장용 조성물을 제공하는 것이다.In addition, it is to provide a dressing composition comprising (essentially) the strain or a culture thereof as an active ingredient.
본 발명의 또 다른 목적은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 생균제 조성물을 제공하는 것이다.Another object of the present invention is to provide a probiotic composition comprising the strain or a culture thereof as an active ingredient.
또한 상기 균주 또는 이의 배양물을 유효성분으로 (필수적으로) 이루어지는 생균제 조성물을 제공하는 것이다.In addition, it is to provide a probiotic composition (essentially) consisting of the strain or a culture thereof as an active ingredient.
본 발명의 또 다른 목적은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 면역조절용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for immunomodulation comprising the strain or a culture thereof as an active ingredient.
또한 상기 균주 또는 이의 배양물을 유효성분으로 (필수적으로) 이루어지는 면역조절용 조성물을 제공하는 것이다.In addition, it is to provide a composition for immunomodulation (essentially) consisting of the strain or a culture thereof as an active ingredient.
본 발명의 또 다른 목적은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 식품첨가용 조성물을 제공하는 것이다.Another object of the present invention is to provide a food additive composition comprising the strain or a culture thereof as an active ingredient.
또한 상기 균주 또는 이의 배양물을 유효성분으로 (필수적으로) 이루어지는 식품첨가용 조성물을 제공하는 것이다.In addition, it is to provide a composition for food additives (essentially) composed of the above-described strain or a culture thereof.
본 발명의 또 다른 목적은 염증성 질환의 예방 또는 치료용 제제를 제조하기 위한 상기 균주 또는 이의 배양물의 용도를 제공하는 것이다.Another object of the present invention is to provide the use of the strain or its culture for preparing an agent for the prevention or treatment of inflammatory diseases.
본 발명의 또 다른 목적은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 조성물의 유효량을 이를 필요로 하는 개체에 투여하는 것을 포함하는 염증성 질환의 치료 방법을 제공하는 것이다.Another object of the present invention is to provide a method of treating an inflammatory disease, comprising administering an effective amount of a composition comprising the strain or a culture thereof as an active ingredient to an individual in need thereof.
본 발명의 또 다른 목적은 생균제를 제조하기 위한 상기 균주 또는 이의 배양물의 용도를 제공하는 것이다.Another object of the present invention is to provide a use of the strain or its culture for producing a probiotic.
본 발명의 또 다른 목적은 정장용 제제를 제조하기 위한 상기 균주 또는 이의 배양물의 용도를 제공하는 것이다.Another object of the present invention is to provide the use of the strain or its culture for preparing a preparation for dressing.
본 발명의 또 다른 목적은 면역조절용 제제를 제조하기 위한 상기 균주 또는 이의 배양물의 용도를 제공하는 것이다.Another object of the present invention is to provide the use of the strain or its culture for preparing an agent for immunomodulation.
본 발명의 또 다른 목적은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 조성물의 유효량을 이를 필요로 하는 개체에 투여하는 것을 포함하는 면역조절의 방법을 제공하는 것이다.Another object of the present invention is to provide a method of immunomodulation comprising administering an effective amount of a composition comprising the strain or a culture thereof as an active ingredient to an individual in need thereof.
본 발명의 또 다른 목적은 식품첨가용 제제를 제조하기 위한 상기 균주 또는 이의 배양물의 용도를 제공하는 것이다.Another object of the present invention is to provide a use of the strain or a culture thereof for preparing a food additive preparation.
상기와 같은 목적을 달성하기 위하여, 본 발명은 카자츠타니아 투리센시스(kazachstania turicensis) CAU Y1706(수탁번호 : KCTC13794BP) 균주를 제공한다.In order to achieve the above object, the present invention provides a strain of Kazachstania turicensis CAU Y1706 (Accession No.: KCTC13794BP).
본 발명의 다른 목적을 달성하기 위하여, 본 발명은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 약학적 조성물을 제공한다.In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising the strain or a culture thereof as an active ingredient.
또한 본 발명은 상기 균주 또는 이의 배양물을 유효성분으로 (필수적으로) 이루어지는 염증성 질환의 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising (essentially) the strain or a culture thereof as an active ingredient.
본 발명의 다른 목적을 달성하기 위하여, 본 발명은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 염증성 질환의 예방 또는 개선용 식품 조성물을 제공한다. In order to achieve another object of the present invention, the present invention provides a food composition for preventing or improving inflammatory diseases comprising the strain or a culture thereof as an active ingredient.
또한 본 발명은 상기 균주 또는 이의 배양물을 유효성분으로 (필수적으로) 이루어지는 염증성 질환의 예방 또는 개선용 식품 조성물을 제공한다.In addition, the present invention provides a food composition for preventing or improving inflammatory diseases comprising (essentially) the strain or its culture as an active ingredient.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 염증성 질환의 예방 또는 개선용 화장료 조성물을 제공한다.In order to achieve another object of the present invention, the present invention provides a cosmetic composition for preventing or improving inflammatory diseases comprising the strain or a culture thereof as an active ingredient.
또한 본 발명은 상기 균주 또는 이의 배양물을 유효성분으로 (필수적으로) 이루어지는 염증성 질환의 예방 또는 개선용 화장료 조성물을 제공한다.In addition, the present invention provides a cosmetic composition for preventing or improving inflammatory diseases comprising (essentially) the strain or its culture as an active ingredient.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 정장용 조성물을 제공한다.In order to achieve another object of the present invention, the present invention provides a dressing composition comprising the strain or a culture thereof as an active ingredient.
또한 본 발명은 상기 균주 또는 이의 배양물을 유효성분으로 (필수적으로) 이루어지는 정장용 조성물을 제공한다.In addition, the present invention provides a dressing composition comprising (essentially) the strain or a culture thereof as an active ingredient.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 생균제 조성물을 제공한다.In order to achieve another object of the present invention, the present invention provides a probiotic composition comprising the strain or a culture thereof as an active ingredient.
또한 본 발명은 상기 균주 또는 이의 배양물을 유효성분으로 (필수적으로) 이루어지는 생균제 조성물을 제공한다.In addition, the present invention provides a probiotic composition (essentially) comprising the strain or a culture thereof as an active ingredient.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 면역조절용 조성물을 제공한다.In order to achieve another object of the present invention, the present invention provides a composition for immunomodulation comprising the strain or a culture thereof as an active ingredient.
또한 본 발명은 상기 균주 또는 이의 배양물을 유효성분으로 (필수적으로) 이루어지는 면역조절용 조성물을 제공한다.In addition, the present invention provides a composition for immunomodulation comprising (essentially) the strain or a culture thereof as an active ingredient.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 식품첨가용 조성물을 제공한다.In order to achieve another object of the present invention, the present invention provides a food additive composition comprising the strain or its culture as an active ingredient.
또한 본 발명은 상기 균주 또는 이의 배양물을 유효성분으로 (필수적으로) 이루어지는 식품첨가용 조성물을 제공한다.In addition, the present invention provides a composition for food additives (essentially) comprising the strain or a culture thereof as an active ingredient.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 염증성 질환의 예방 또는 치료용 제제를 제조하기 위한 상기 균주 또는 이의 배양물의 용도를 제공한다.In order to achieve another object of the present invention, the present invention provides the use of the strain or its culture for preparing a preparation for the prevention or treatment of inflammatory diseases.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 조성물의 유효량을 이를 필요로 하는 개체에 투여하는 것을 포함하는 염증성 질환의 치료 방법을 제공한다.In order to achieve another object of the present invention, the present invention provides a method of treating an inflammatory disease comprising administering an effective amount of a composition comprising the strain or a culture thereof as an active ingredient to an individual in need thereof.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 생균제를 제조하기 위한 제1항의 균주 또는 이의 배양물의 용도를 제공한다.In order to achieve another object of the present invention, the present invention provides the use of the strain of claim 1 or a culture thereof for producing a probiotic.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 정장용 제제를 제조하기 위한 상기 균주 또는 이의 배양물의 용도를 제공한다.In order to achieve another object of the present invention, the present invention provides the use of the strain or a culture thereof for preparing a preparation for dressing.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 면역조절용 제제를 제조하기 위한 상기 균주 또는 이의 배양물의 용도를 제공한다.In order to achieve another object of the present invention, the present invention provides the use of the strain or its culture for preparing an agent for immunomodulation.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 조성물의 유효량을 이를 필요로 하는 개체에 투여하는 것을 포함하는 면역조절의 방법을 제공한다.In order to achieve another object of the present invention, the present invention provides a method of immunomodulation comprising administering an effective amount of a composition comprising the strain or a culture thereof as an active ingredient to an individual in need thereof.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 식품첨가용 제제를 제조하기 위한 상기 균주 또는 이의 배양물의 용도를 제공한다.In order to achieve another object of the present invention, the present invention provides the use of the strain or its culture for preparing a food additive preparation.
이하 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
본 발명은 The present invention 카자츠타니아Kazakhstan 투리센시스Turismensis (( kazachstaniakazachstania turicensisturicensis ) ) CAUCAU Y1706(수탁번호 : Y1706 (Accession number: KCTC13794BPKCTC13794BP ) 균주를 제공한다.) Strain.
상기 카자츠타니아 투리센시스는 항염증 활성을 갖는 것을 특징으로 하는 균주로서, 특히 Th1 사이토카인 수치는 증가시키면서, Th2 사이토카인 수치는 감소시킴에 따라 Th1/Th2 면역반응의 불균형, 특히 Th2 면역반응 과잉의 면역반응 불균형을 개선하는 것을 특징으로 한다.The Kazaztania thuricensis is a strain characterized by having anti-inflammatory activity. In particular, while increasing the Th1 cytokine level and decreasing the Th2 cytokine level, the imbalance of the Th1/Th2 immune response, in particular the Th2 immune response It is characterized by improving the imbalance of the excess immune response.
본 발명자들은 김치에서 본 발명의 균주를 분리하여, IL-4, IL5와 같은 Th2 사이토카인의 농도를 유의미하게 감소시키며, IFN-γ, TNF-α, IL-12와 같은 Th1 사이토카인의 수치는 증가시키고, Treg 사이토카인 수치 또한 감소시킴으로써 Th2 사이토카인을 억제하는 신규한 효모 카자츠타니아 투리센시스 CAU Y1706을 선별하였다. 선별된 균주는 18s, 26s rRNA 염기서열을 이용하여 분자생물학적으로 분석한 결과, 신규한 카자츠타니아 투리센시스 효모인 것으로 확인하고, CAU Y1706으로 명명하였다. 본 균주는 한국생명공학연구원 생물자원센터(KCTC)에 2019년 01월 22일 기탁되었다(수탁번호 : KCTC13794BP). The present inventors isolate the strain of the present invention from kimchi, significantly reduce the concentration of Th2 cytokines such as IL-4 and IL5, and the levels of Th1 cytokines such as IFN-γ, TNF-α, IL-12 are A novel yeast Kazaztania thuricensis CAU Y1706 that inhibits Th2 cytokines was selected by increasing and also reducing Treg cytokine levels. The selected strain was analyzed by molecular biology using 18s, 26s rRNA sequencing, and confirmed to be a novel Kazaztania thuricensis yeast, and named as CAU Y1706. This strain was deposited on January 22, 2019 at the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center (KCTC) (Accession No.: KCTC13794BP).
본 발명에 따른 카자츠타니아 투리센시스(kazachstania turicensis)는 종래에는 saccharomyces turicensis로 불리며, 건강 발효유 kefir grain에 존재하는 효모로 알려져있다. Kazachstania turicensis according to the present invention is conventionally called saccharomyces turicensi s, and is known as yeast present in kefir grains of healthy fermented milk.
본 발명의 실시예에 따르면, 본 발명의 신규 효모는 면역사이토카인 조절 뿐만 아니라, 혈청 내 IgE, 호산구, 호중구, 호염구를 감소시키고, 장내 미생물 균총 또한 조절하는 것을 확인하였다.According to an embodiment of the present invention, it was confirmed that the novel yeast of the present invention not only regulates immunocytokines, but also reduces IgE, eosinophils, neutrophils, basophils in serum, and also regulates microbial flora in the intestine.
본 발명의 또 다른 실시예에 따르면, 본 발명의 신규 효모는 오브알부민 감작 마우스를 이용한 모델에 대하여 피부 및 장에서 염증 개선효과를 나타내었다. 구체적으로, 등 피부세포 및 회장 병변의 비만세포의 축적을 억제하며 호산구 수 또한 감소시키는 것이 확인되었다. 뿐만 아니라 등 피부의 호산구 침윤 수준 또한 감소시키는 것을 확인하였다. According to another embodiment of the present invention, the novel yeast of the present invention showed an effect of improving inflammation in skin and intestine in a model using ovalbumin-sensitized mice. Specifically, it was confirmed that it suppresses the accumulation of mast cells in back skin cells and ileal lesions and also reduces eosinophil counts. In addition, it was confirmed that the eosinophil infiltration level of the back skin was also reduced.
이에 더하여, 본 발명에 따른 신규 효모는 내산성 및 내담즙성 또한 우수한 것으로 확인되었다.In addition, it was confirmed that the novel yeast according to the present invention is also excellent in acid resistance and bile resistance.
본 발명의 실시예에서는 신규 효모의 내산성을 확인하기 위하여 pH 2.0 조건에서 실험을 진행하였으나, 본 발명의 효모 균수가 시간이 지날수록 상당한 속도로 증가하였는 바, 본 발명의 효모가 강산의 조건에서 저해되지 않음을 확인하였다. 뿐만 아니라, 담즙산 0.2% 및 0.4% 조건에서도 균이 우수한 성장 능력을 나타내었다.In the embodiment of the present invention, experiments were performed under the condition of pH 2.0 to confirm the acid resistance of the new yeast, but the number of yeast bacteria of the present invention increased at a significant rate over time, and the yeast of the present invention was inhibited under the condition of strong acid Was confirmed. In addition, the bacteria showed excellent growth ability even under the condition of 0.2% and 0.4% of bile acids.
요컨대 본 발명에 따른 신규 효모는 항염증, 특히 피부 염증을 감소시키는 효과가 우수할 뿐만 아니라, 정장 효과가 있으며, 내산성 및 내담즙성이 우수하여 우수하게 장내에 도달할 수 있으므로 상업적 이용가치가 높다.In short, the new yeast according to the present invention is not only excellent in anti-inflammatory effect, particularly in reducing skin inflammation, but also has a suit effect, and has excellent acid resistance and bile resistance, so it can reach the intestine excellently, so its commercial use value is high. .
이에 따라 본 발명은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 약학적 조성물을 제공한다. Accordingly, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising the strain or a culture thereof as an active ingredient .
본 명세서에서 용어 "을 포함하는(comprising)"이란 "함유하는(including)"또는 "특징으로 하는(characterized by)"과 동일한 의미로 사용되며, 본 발명에 따른 조성물 또는 방법에 있어서, 구체적으로 언급되지 않은 추가적인 구성 성분 또는 방법의 단계 등을 배제하지 않는다. 또한 용어 "로 이루어지는(consisting of)"이란 별도로 기재되지 않은 추가적인 요소, 단계 또는 성분 등을 제외하는 것을 의미한다. 용어 "필수적으로 이루어지는(essentially consisting of)"이란 조성물 또는 방법의 범위에 있어서, 기재된 물질 또는 단계와 더불어 이의 기본적인 특성에 실질적으로 영향을 미치지 않는 물질 또는 단계 등을 포함할 수 있는 것을 의미한다.As used herein, the term "comprising" is used in the same sense as "including" or "characterized by," and refers specifically to the composition or method according to the present invention. Do not exclude additional components or method steps that have not been made. In addition, the term "consisting of" means excluding additional elements, steps or components, which are not described separately. The term "essentially consisting of" means that in the scope of a composition or method, it may include substances or steps that do not materially affect the basic properties thereof, in addition to the materials or steps described.
본 발명에 따른 '카자츠타니아 투리센시스(kazachstania turicensis) CAU Y1706'또는 '카자츠타니아 투리센시스(kazachstania turicensis) CAU Y1706 균체'는 배양 배지(medium)로부터 분리 또는 정제된 생균 그 자체 뿐 아니라, 당업자에게 알려진 유산균의 임의의 가공형태(특히 생균의 가공 형태)를 포함하는 것으로, 예를 들어 건조물, 동결물 등을 포함하며 그 형태에 제한되지 않는다. 또한 배양 배지에서 상기 균주와 화합하기 쉬운 하나 이상의 화합물과 혼합되거나, 혼합되지 않은 형태로도 이용할 수 있다. According to the present invention, the'kazachstania turicensis CAU Y1706' or the'kazachstania turicensis CAU Y1706 cell' is not only the live cells that have been separated or purified from the culture medium, but also , Which includes any processing form of lactic acid bacteria known to those skilled in the art (especially processing form of live cells), and includes, for example, dried materials, frozen materials, and the like. In addition, it may be used in a culture medium mixed with one or more compounds that are easily compounded with the strain, or in a non-mixed form.
또한 본 발명에 따른 균주는 살아있는 형태, 준-활성(semi-active) 형태, 불활성화된 형태, 또는 사멸된 형태로도 이용될 수 있으나, 살아있는 형태인 것이 바람직하다. 본 발명에 따른 균주를 사멸된 형태로 이용할 때에는 당업계에 알려진 유산균 사균화 방법에 의한 것이라면 제한 없이 선택하여 이용할 수 있다. In addition, the strain according to the present invention may be used in a living form, a semi-active form, an inactivated form, or a killed form, but is preferably a living form. When using the strain according to the present invention in a killed form, it can be selected and used without limitation as long as it is by a method for lactic acid bacteria fertilization known in the art.
본 발명에서 '배양'이란, 미생물을 적절하게 인공적으로 조절한 환경에서 생육시키기 위하여 수행하는 모든 행위를 의미하며, 본 발명에서는 '발효'를 포함하는 개념으로서, '배양물'은 '발효물'을 포함하는 의미이고, 배지에서 배양한 배양액 자체(즉, 균을 포함하고 있는 배양액 자체), 또는 상기 배양액에 열처리 등의 가공을 가한 결과물 등 배양액 자체로부터 파생되는 가공물을 포함한다. 'Cultivation' in the present invention means all actions performed in order to grow microorganisms in an artificially controlled environment, and in the present invention, the concept includes'fermentation', and'culture' is'fermentation'. It includes a mean, and includes a culture medium cultured in the medium itself (that is, the culture medium itself containing bacteria), or a product derived from the culture medium itself, such as a result of processing such as heat treatment to the culture medium.
또한 본 발명에서 염증성 질환은 Th1 매개 면역질환 또는 Th2 매개 면역질환으로서, 보다 구체적으로는 급성 또는 만성 장기이식 거부반응, 이식편대숙주병, 염증성 장 질환, 크론병, 궤양성 대장염, 염증성 피부 질환, 다발성 경화증, 췌장염, 외상 유발 쇼크, 기관지 천식, 알러지성 비염, 알러지성 결막염, 낭포성 섬유증, 급성 기관지염, 만성 기관지염, 급성 세기관지염, 만성 세기관지염, 골관절염, 통풍, 척추관절병증, 강직성 척추염, 라이터 증후군, 건선성 관절병증, 장질환 척추염, 연소자성 관절병증, 연소자성 강직성 척추염, 반응성 관절병증, 감염성 관절염, 후-감염성 관절염, 루게릭병, 결절성 다발동맥염, 과민성 혈관염, 루게릭 육아종증, 류마티스성 다발성 근육통, 관절세포 동맥염, 칼슘 결정침착관절병증, 가성 통풍, 비-관절 류마티즘, 점액낭염, 건초염, 상과염, 신경병증성 관절질환(neuropathic joint disease 또는 charcot joint), 출혈성 관절증(hemarthrosis), 알레르기 자반병, 비후성 골관절병증, 다중심성 세망조직구종, 척추측만증(scoliosis), 혈색소증, 혈색소병증, 고지단백혈증, 저감마글로불린혈증, 가족성 지중해열(familial Mediterranean fever), 베체트씨병, 전신성 홍반성 루푸스, 재귀열, 패혈증, 패혈성 쇼크, 급성 호흡곤란 증후군, 다발성 장기부전, 만성 폐쇄성 폐질환(chronic obstructive pulmonary disease), 류마티스성 관절염(rheumatoid arthritis), 급성 폐손상(acute lung injury), 기관지 폐 형성장애(broncho-pulmonary dysplasia), 제1형 당뇨병, 제2형 당뇨병, 동맥경화, 알츠하이머성 치매, 가족성 한랭자가염증 증훈군(familial cold autoinflammatory syndrome), 머클-웰스 증후군(Muckle-Wells syndrome), 신생아발병 다발성 염증성 질환(neonatal multisystem inflammatory disease), 만성 유아 신경피부관절 증후군(chronic infantile neurologic cutaneous articular syndrome), 성인발증형 스틸병(adult-onset Still's disease), 접촉성 피부염, 포상기태(hydatidiform mole), PAPA 증후군(syndrome of pyogenic arthritis, pyoderma gangrenosum and acne), 고면역글로불린 D 증후군(hyperimmunoglobulin d syndrome), 크리오피린 관련 주기적 증후군(cryopyrin-associated periodic syndrome), 각막염, 결막염, 망막염, 망막혈관염, 포도막염, 안검염, 알레르기결막염, 건성 안(dry eye), 전신성 경피증(progressive systemic sclerosis), 다발성 근염(polymyositis), 자가면역성 뇌척수염, 중증 근무력증(myasthenia gravis), 결절성 다발성 동맥염(polyarteritis nodosa) 및 섬유조직염(fibromyalgia syndrome)로 이루어진 군에서 선택되는 어느 하나 이상의 질환인 것일 수 있다.In addition, the inflammatory disease in the present invention is a Th1-mediated immune disease or Th2-mediated immune disease, more specifically, acute or chronic organ transplant rejection, graft versus host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, inflammatory skin disease, Multiple sclerosis, pancreatitis, traumatic shock, bronchial asthma, allergic rhinitis, allergic conjunctivitis, cystic fibrosis, acute bronchitis, chronic bronchitis, acute bronchiolitis, chronic bronchiolitis, osteoarthritis, gout, spondylosis, ankylosing spondylitis, Reiter syndrome, Psoriatic arthrosis, bowel disease spondylitis, combustive arthrosis, combustive ankylosing spondylitis, reactive arthritis, infectious arthritis, post-infectious arthritis, Lou Gehrig's disease, nodular polyarteritis, irritable vasculitis, Lou Gehrig's granulomatosis, rheumatoid multiple myalgia, Arthritis arthritis, calcium degenerative arthritis, pseudogout, non-articular rheumatism, mucositis, hayitis, epicondylitis, neuropathic joint disease or charcot joint, hemorrhagic joint, allergic purpura, hypertrophic osteoarthritis Pathology, multiplicity reticuloma, scoliosis, hemochromatosis, hemoglobinemia, hyperlipoproteinemia, hypomaglobulinemia, familial Mediterranean fever, Behcet's disease, systemic lupus erythematosus, recurrence fever, sepsis, Septic shock, acute respiratory distress syndrome, multiple organ failure, chronic obstructive pulmonary disease, rheumatoid arthritis, acute lung injury, broncho-pulmonary dysplasia ), type 1 diabetes, type 2 diabetes, arteriosclerosis, Alzheimer's dementia, familial cold autoinflammatory syndrome, Muckle-Wells syndrome, multiple inflammatory diseases of neonatal disease ( ne onatal multisystem inflammatory disease, chronic infantile neurologic cutaneous articular syndrome, adult-onset still's disease, contact dermatitis, hydatidiform mole, syndrome of PAPA pyogenic arthritis, pyoderma gangrenosum and acne, hyperimmunoglobulin d syndrome, cryopyrin-associated periodic syndrome, keratitis, conjunctivitis, retinitis, retinitis, uveitis, blepharitis, allergic conjunctivitis, dryness Choose from the group consisting of dry eye, progressive systemic sclerosis, polymyositis, autoimmune encephalomyelitis, myasthenia gravis, polyarteritis nodosa, and fibromyalgia syndrome. It may be any one or more diseases.
본 명세서에서 "장기이식 거부반응(transplant rejection)"이란, 구체적으로는 심장, 폐, 심장 및 폐 복합, 간, 신장, 췌장, 피부, 장(bowel) 또는 각막 등 고형장기의 이식 후에 이식받은 환자의 체내 면역세포가 이식기관에 침윤하고 공격함으로써 세포사멸과 조직 괴사 등을 유발하여 발생하는 급성 또는 만성의 이식 거부반응, 그리고 골수이식 후 이식편대숙주병(graft-versus-host disease, GVHD)일 수 있다.As used herein, "transplant rejection" refers to a patient transplanted after transplantation of a solid organ such as a heart, lung, heart and lung complex, liver, kidney, pancreas, skin, bowel, or cornea. Acute or chronic transplant rejection, caused by cell death and tissue necrosis by invading and attacking immune cells in the body, and graft-versus-host disease (GVHD) after bone marrow transplantation Can.
상기 염증성 질환 중 염증성 피부 질환은 건선, 아토피성 피부염, 습진성 피부염, 접촉성 피부염, 지루성 피부염, 장미색 비강진, 편평 태선, 혈관염, 모공성 홍색 비강진, 봉와직염, 모낭염, 옹종, 천포창, 수포성(bullous) 천포창, 표피 수포증, 담마진, 혈관 부종, 맥관염, 홍반 및 피부 호산구 증가증으로 이루어진 군에서 선택되는 하나 이상의 질환인 것을 특징으로 한다.Among the inflammatory diseases, inflammatory skin diseases include psoriasis, atopic dermatitis, eczematous dermatitis, contact dermatitis, seborrheic dermatitis, rosaceous nasal stiffness, lichen planus, vasculitis, pore red nasal hypertension, cellulitis, folliculitis, carcinoma, pemphigus, bullous ) It is characterized by one or more diseases selected from the group consisting of celestial swelling, epidermal vesicles, dammajin, angioedema, vasculitis, erythema and skin eosinophilia.
본 발명에서의 용어, "예방"이란 본 발명에 따른 약학 조성물의 투여에 의해 퇴행성 뇌질환의 발병을 억제 또는 지연시키는 모든 행위를 의미하고, "치료"란 상기 약학 조성물의 투여에 의해 퇴행성 뇌질환의 의심 및 발병 개체의 증상이 호전되거나 이롭게 변경되는 모든 행위를 의미한다.The term "prevention" in the present invention means all actions to suppress or delay the development of degenerative brain disease by administration of the pharmaceutical composition according to the present invention, and "treatment" means degenerative brain disease by administration of the pharmaceutical composition. It means any action that the symptoms of the suspected and affected individual are improved or beneficially altered.
본 발명에서 상기 조성물은 사용목적 내지 양상에 따라 함량은 크게 제한되지 않으며, 예를 들면 조성물 총 중량을 기준으로 0.01~99 중량%, 바람직하게는 0.5~50 중량%, 더 바람직하게는 1~30 중량%일 수 있다. 또한, 본 발명에 따른 약학 조성물은 유효성분 외에 약학적으로 허용가능한 담체, 부형제 또는 희석제와 같은 첨가제를 더 포함할 수 있다. In the present invention, the content of the composition is not greatly limited depending on the purpose or aspect of use, for example, 0.01 to 99% by weight, preferably 0.5 to 50% by weight, more preferably 1 to 30 based on the total weight of the composition. Weight percent. In addition, the pharmaceutical composition according to the present invention may further include an additive such as a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient.
본 발명에 따른 약학적 조성물은 약학적으로 유효한 양의 상기 균주를 단독으로 포함하거나 하나 이상의 약학적으로 허용되는 담체를 추가로 포함할 수 있다. 본 발명의 약학적 조성물은 단일 투여량(single dose)으로 환자에게 투여될 수 있으며, 다중 투여량(multiple dose)이 장기간 투여되는 분할 치료방법(fractionated treatment protocol)에 의해 투여될 수 있다. 상기에서 약학적으로 유효한 양이란 음성 대조군에 비해 그 이상의 반응을 나타내는 양을 말하며 바람직하게는 퇴행성 뇌질환을 치료 또는 예방하기에 충분한 양을 말한다. 본 발명에 따른 균주의 유효한 양으로는 0.001 내지 1000 mg/kg b.w./day일 수 있고, 바람직하게 1 내지 2000mg/kg b.w./day일 수 있으나 이에 한정되는 것은 아니다. 그러나 상기 약학적으로 유효한 양은 질환 및 이의 중증정도, 환자의 연령, 체중, 건강상태, 성별, 투여 경로 및 치료기간 등과 같은 여러 인자에 따라 적절히 변화될 수 있다.The pharmaceutical composition according to the present invention may contain a pharmaceutically effective amount of the strain alone or may further include one or more pharmaceutically acceptable carriers. The pharmaceutical composition of the present invention may be administered to a patient in a single dose, and may be administered by a fractionated treatment protocol in which multiple doses are administered for a long time. The pharmaceutically effective amount in the above refers to an amount that exhibits a higher response than the negative control group, and preferably refers to an amount sufficient to treat or prevent degenerative brain disease. The effective amount of the strain according to the present invention may be 0.001 to 1000 mg/kg b.w./day, preferably 1 to 2000 mg/kg b.w./day, but is not limited thereto. However, the pharmaceutically effective amount may be appropriately changed according to various factors such as the disease and its severity, the patient's age, weight, health status, sex, administration route, and treatment period.
본 발명의 조성물은 상기 약학적으로 허용되는 담체와 함께 당업계에 공지된 방법으로 투여경로에 따라 다양하게 제형화될 수 있다. The composition of the present invention can be variously formulated according to the route of administration by a method known in the art together with the pharmaceutically acceptable carrier.
본 발명의 약학 조성물은 목적하는 방법에 따라 인간을 포함한 포유류에 경구 투여되거나 비경구 투여될 수 있으며, 비경구 투여 방식으로는 피부 외용, 복강내주사, 직장내주사, 피하주사, 정맥주사, 근육내 주사 또는 흉부내 주사 주입방식 등이 있다. The pharmaceutical composition of the present invention may be administered orally or parenterally to mammals, including humans, according to the desired method, and the parenteral administration methods include external application to the skin, intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, and muscle. Intra-injection or intra-thoracic injection.
상기에서 "약학적으로 허용되는" 이란 생리학적으로 허용되고 인간에게 투여될 때, 활성 성분의 작용을 저해하지 않으며 통상적으로 위장 장애, 현기증과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 비독성의 조성물을 말한다. 본 발명의 조성물은 상기 약학적으로 허용되는 담체와 함께 당업계에 공지된 방법으로 투여경로에 따라 다양하게 제형화될 수 있다. 투여 경로로는 이에 한정되지는 않으나 경구적 또는 비경구적으로 투여될 수 있고, 바람직하게는 경구투여 일 수 있다."Pharmaceutically acceptable" as used herein is a non-toxic composition that is physiologically acceptable and does not inhibit the action of the active ingredient when administered to humans and usually does not cause allergic or similar reactions such as gastrointestinal disorders, dizziness, or the like. Says The composition of the present invention can be variously formulated according to the route of administration by a method known in the art together with the pharmaceutically acceptable carrier. The route of administration is not limited to this, but may be administered orally or parenterally, preferably oral administration.
본 발명의 약학적 조성물을 경구 투여하는 경우 본 발명의 약학적 조성물은 적합한 경구 투여용 담체와 함께 당업계에 공지된 방법에 따라 분말, 과립, 정제, 환제, 당의정제, 캡슐제, 액제, 겔제, 시럽제, 현탁액, 웨이퍼 등의 형태로 제형화될 수 있다. 예를 들어, 경구용 제제는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 활성성분을 고체 부형제와 배합한 다음 이를 분쇄하고 적합한 보조제를 첨가한 후 과립 혼합물로 가공함으로써 정제 또는 당의정제를 수득할 수 있다. 적합한 부형제의 예로는 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 및 말티톨 등을 포함하는 당류와 옥수수 전분, 밀 전분, 쌀 전분 및 감자 전분 등을 포함하는 전분류, 셀룰로즈, 메틸 셀룰로즈, 나트륨 카르복시메틸셀룰로오즈 및 하이드록시프로필메틸-셀룰로즈 등을 포함하는 셀룰로즈류, 젤라틴, 폴리비닐피롤리돈 등과 같은 충전제가 포함될 수 있다. 또한, 경우에 따라 가교결합 폴리비닐피롤리돈, 한천, 알긴산 또는 나트륨 알기네이트 등을 붕해제로 첨가할 수 있다. 나아가, 본 발명의 약학적 조성물은 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다.When the pharmaceutical composition of the present invention is administered orally, the pharmaceutical composition of the present invention is a powder, granules, tablets, pills, dragees, capsules, liquids, gels according to methods known in the art together with a suitable carrier for oral administration. , Syrups, suspensions, wafers, and the like. For example, oral preparations include tablets, pills, powders, granules, capsules, and the like, and the active ingredient is combined with a solid excipient, then crushed and added with a suitable adjuvant, and then processed into a granule mixture to tablet or dragee tablets. Can be obtained. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol, and starches including corn starch, wheat starch, rice starch, potato starch, and the like. Fillers such as cellulose, gelatin, polyvinylpyrrolidone, etc. may be included, including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose. In addition, if necessary, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may further include an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and a preservative.
또한, 비경구적으로 투여하는 경우 본 발명의 약학적 조성물은 적합한 비경구용 담체와 함께 당 업계에 공지된 방법에 따라 제형화될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함될 수 있다. 비수성용제, 현탁용제로는 프로필렌글리콜(Propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 그 밖의 약학적으로 허용되는 담체로는 당업계에 공지되어 있는 것을 참고로 할 수 있다.In addition, when administered parenterally, the pharmaceutical composition of the present invention may be formulated according to methods known in the art together with a suitable parenteral carrier. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As other pharmaceutically acceptable carriers, reference may be made to those known in the art.
좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로 제라틴 등이 사용될 수 있다. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.
나아가, 본 발명의 약학적 조성물은 퇴행성 뇌질환을 예방 및 치료하는 효과를 가지는 공지의 화합물과 병행하여 투여할 수 있다.Furthermore, the pharmaceutical composition of the present invention can be administered in parallel with a known compound having an effect of preventing and treating degenerative brain disease.
더 나아가 당 분야의 적정한 방법으로 또는 Remington's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다. Furthermore, it can be preferably formulated in accordance with each disease or ingredient using methods disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA, or by appropriate methods in the art.
또한, 본 발명은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 염증성 질환의 예방 또는 개선용 화장료 조성물을 제공한다.In addition, the present invention provides a cosmetic composition for preventing or improving inflammatory diseases comprising the strain or a culture thereof as an active ingredient.
본 발명에 따른 화장료 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 본 발명에 따른 신규 균주 또는 이의 배양물 이외에 피부과학적으로 허용 가능한 매질, 기제, 보조제, 부형제 등과 함께 피부과학 분야에서 통상적으로 제조되는 기초 화장품 조성물(화장수, 크림, 에센스, 클렌징폼 및 클렌징 워터와 같은 세안제, 팩, 바디오일, 미스트, 비누), 색조 화장품 조성물(파운데이션, 립스틱, 마스카라, 메이크업 베이스), 두발 제품 조성물(샴푸, 린스, 헤어컨디셔너, 헤어젤) 및 비누 등의 형태로 제조될 수 있다.The cosmetic composition according to the present invention may be prepared in any formulation conventionally prepared in the art, and dermatology together with a dermatologically acceptable medium, base, adjuvant, excipient, etc. in addition to the novel strain or culture thereof according to the present invention Basic cosmetic compositions commonly produced in the field (face washes, creams, essences, cleansing agents such as cleansing foams and cleansing water, packs, body oils, mists, soaps), color cosmetic compositions (foundation, lipstick, mascara, makeup base), hair Product composition (shampoo, conditioner, hair conditioner, hair gel) and soap may be prepared.
상기 부형제로는 이에 한정되지는 않으나 예를 들어, 피부연화제, 피부 침투 증강제, 착색제, 방향제, 유화제, 연화제, 농화제, 농축제, 겔화제, 현택화제, 안정화제, 발포제(foaming agent), 계면활성제, 충전제 또는 용매를 포함할 수 있다. 또한 향료, 색소, 살균제, 산화방지제, 방부제, 보조제 및 보습제 등을 추가로 포함할 수 있으며, 물성 개선을 목적으로 점증제, 무기염류, 합성 고분자 물질 등을 포함할 수 있다. 예를 들면, 본 발명의 화장료 조성물로 세안제 및 비누를 제조하는 경우에는 통상의 세안제 및 비누 베이스에 본 발명의 추출물을 첨가하여 용이하게 제조할 수 있다. 크림을 제조하는 경우에는 일반적인 수중유적형(O/W)의 크림베이스에 본 발명의 균주 또는 그 배양물을 첨가하여 제조할 수 있다. 여기에 향료, 킬레이트제, 색소, 산화방지제, 방부제 등과 물성 개선을 목적으로 한 단백질, 미네랄, 비타민 등 합성 또는 천연소재를 추가로 첨가할 수 있다.The excipients are not limited to, for example, skin emollients, skin penetration enhancers, colorants, fragrances, emulsifiers, emollients, thickeners, thickeners, gelling agents, suspending agents, stabilizers, foaming agents, interfaces Active agents, fillers, or solvents. In addition, fragrances, pigments, fungicides, antioxidants, preservatives, auxiliary agents and moisturizers may be further included, and for the purpose of improving physical properties, may include thickeners, inorganic salts, synthetic polymer materials, and the like. For example, in the case of preparing the cleanser and soap with the cosmetic composition of the present invention, it can be easily prepared by adding the extract of the present invention to a conventional cleanser and soap base. When preparing a cream, it can be prepared by adding the strain of the present invention or a culture thereof to a cream base of a general oil-in-water type (O/W). Synthetic or natural materials such as proteins, minerals, vitamins, etc. for the purpose of improving physical properties may be additionally added to flavors, chelating agents, pigments, antioxidants, preservatives, and the like.
적합한 화장료 조성물의 제형으로는 예를 들면 용액, 겔, 고체 또는 반죽 무수 생성물, 수상에 유상을 분산시켜 얻은 에멀젼, 현탁액, 마이크로에멀젼, 마이크로캡슐, 미세과립구 또는 이온형(리포좀), 비이온형의 소낭 분산제의 형태, 크림, 스킨, 로션, 파우더, 연고, 스프레이 또는 콘실 스틱(conceal stick)의 형태로 제공될 수 있다. 또한, 포말(foam)의 형태 또는 압축된 추진제를 더 함유한 에어로졸 조성물의 형태로도 제조될 수 있다. 본 발명의 화장료 조성물을 첨가할 수 있는 제품으로는 이에 한정되는 것은 아니나 스킨로션, 스킨 소프너, 스킨토너, 수렴화장수, 유연화장수, 영양화장수, 아스트린젠트, 로션, 밀크로션, 모이스처 로션, 영양로션, 바디크림, 마사지크림, 영양크림, 모이스처 크림, 핸드크림, 에센스, 영양에센스, 팩, 비누, 샴푸, 클렌징폼, 클렌징로션, 클렌징크림, 바디로션, 바디클렌저, 트리트먼트, 미용액, 유액, 프레스파우더, 루스파우더, 아이섀도 등의 제형을 포함한다.Suitable cosmetic composition formulations include, for example, solutions, gels, solid or dough anhydrous products, emulsions obtained by dispersing an oil phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes), nonionic It may be provided in the form of a vesicle dispersant, cream, skin, lotion, powder, ointment, spray or conceal stick. It can also be prepared in the form of a foam or aerosol composition further containing a compressed propellant. The product to which the cosmetic composition of the present invention can be added is not limited to, but is not limited to, skin lotion, skin softener, skin toner, converging makeup, softening makeup, nutritional makeup, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, body Cream, massage cream, nutrition cream, moisturizing cream, hand cream, essence, nutrition essence, pack, soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, treatment, essence, emulsion, press powder, Formulations such as rust powder, eye shadow, and the like.
또한 본Also seen 발명은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 염증성 질환의 예방 또는 개선용 식품조성물을 제공한다. The present invention provides a food composition for preventing or improving an inflammatory disease comprising the strain or a culture thereof as an active ingredient.
본 발명에 따른 식품용 조성물은 기능성 식품(functional food), 영양 보조제(nutritional supplement), 건강식품(health food), 건강기능식품 및 식품 첨가제(food additives) 등의 모든 형태를 포함한다. 상기 유형들은 당업계에 공지된 통상적인 방법에 따라 다양한 형태로 제조할 수 있다.The food composition according to the present invention includes all forms of functional food, nutritional supplement, health food, health functional food and food additives. These types can be prepared in various forms according to conventional methods known in the art.
예를 들면, 건강식품으로는 본 발명의 식품용 조성물 자체를 차, 쥬스 및 드링크의 형태로 제조하여 음용하도록 하거나, 과립화, 캡슐화 및 분말화하여 섭취할 수 있다. 또한 본 발명의 식품용 조성물은 피부 세포 보호나 주름 개선의 효과가 있다고 알려진 공지의 물질 또는 활성 성분과 함께 혼합하여 조성물의 형태로 제조할 수 있다. For example, as a health food, the food composition itself of the present invention may be prepared in the form of tea, juice, and drink to be consumed or granulated, encapsulated, and powdered. In addition, the food composition of the present invention can be prepared in the form of a composition by mixing with a known substance or active ingredient known to have an effect of protecting skin cells or improving wrinkles.
상기 건강 식품으로 예를 들면, 침출차, 액상차, 음료, 발효유, 치즈, 젤리, 요구르트, 주스, 생균 제제 및 건강보조식품 등으로 가공될 수 있으며, 그 외 목적에 따라 다양한 식품 첨가제의 형태로 사용될 수 있다. 또한, 건강기능식품의 제형은 용액, 유화물, 점성형 혼합물, 타블렛, 분말, 환 등의 다양한 형태일 수 있으며, 이는 단순 음용, 주사투여, 스프레이 방식 또는 스퀴즈 방식 등의 다양한 방법으로 투여될 수 있다.The health food, for example, can be processed into leached tea, liquid tea, beverage, fermented milk, cheese, jelly, yogurt, juice, probiotic preparations, and health supplements, and can be used in the form of various food additives according to other purposes Can. In addition, the formulation of the dietary supplement may be in various forms, such as solutions, emulsions, viscous mixtures, tablets, powders, pills, etc., which may be administered by various methods such as simple drinking, injection, spray or squeeze. .
바람직하게는, 본 발명의 식품 조성물은 이너뷰티 푸드(inner beauty) 형태로 섭취함으로써 더욱 우수한 효과를 갖는 장점을 가진다. 상기 이너뷰티(inner beauty)는 '먹는 화장품 또는 뷰티푸드'로 일컬어지는 푸드로, 피부에 좋은 여러 가지 성분을 몸속으로 흡수시켜 피부 체질을 건강하게 바꾸는 식품을 지칭하며, 피부 타입에 맞는 화장품을 고르듯 피부 컨디션과 라이프스타일을 고려해 개개인에게 맞는 이너뷰티 푸드를 선택하여 섭취할 수 있다. 보다 바람직하게는 상기 화장료 조성물을 포함하는 화장품과 상기 균주를 포함하는 이너뷰티 푸드를 혼용할 경우, 화장품만 사용하는 것에 비해 그 효과가 월등히 높아져 더욱 효과적인 염증성 질환의 개선 효과를 볼 수 있는 장점을 가진다.Preferably, the food composition of the present invention has the advantage of having a more excellent effect by ingesting in the form of an inner beauty food. The inner beauty is a food called'eating cosmetics or beauty food', refers to food that absorbs various ingredients that are good for the skin into the body and changes the skin's constitution to be healthy, and chooses cosmetics that fit the skin type. You can choose the inner beauty food that suits each individual and take it in consideration of the skin condition and lifestyle. More preferably, when the cosmetic composition containing the cosmetic composition and the inner beauty food containing the strain are mixed, the effect is significantly higher than that of using only the cosmetic product, which has the advantage of showing a more effective improvement effect of inflammatory diseases. .
또한 기능성 식품으로는 음료(알콜성 음료 포함), 과실 및 그의 가공식품 (예: 과일 통조림, 병조림, 잼, 마아말레이드 등), 어류, 육류 및 그 가공식품(예: 햄, 소시지콘비이프 등), 빵류 및 면류(예: 우동, 메밀국수, 라면, 스파게티, 마카로니 등), 과즙, 각종 드링크, 쿠키, 엿, 유제품(예: 버터, 치이즈 등), 식용식물유지, 마아가린, 식물성 단백질, 레토르트 식품, 냉동식품, 각종 조미료(예: 된장, 간장, 소스 등) 등에 본 발명의 식품용 조성물을 첨가하여 제조할 수 있다. 본 발명의 식품용 조성물을 식품첨가제의 형태로 사용하기 위해서는 분말 또는 농축액 형태로 제조하여 사용할 수 있다.In addition, functional foods include beverages (including alcoholic beverages), fruits and processed foods (e.g., canned fruits, canned foods, jams, marmalades, etc.), fish, meat, and processed foods (e.g. ham, sausage corn beef, etc.) ), breads and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), juice, various drinks, cookies, syrup, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, Retort food, frozen food, various seasonings (eg, miso, soy sauce, sauce, etc.) can be prepared by adding the food composition of the present invention. In order to use the food composition of the present invention in the form of a food additive, it may be prepared and used in powder or concentrate form.
상기 본 발명에 따른 식품 조성물에서 유효성분인 본 발명의 균주 함량은 조성물 총 중량을 기준으로 0.01~99 중량%, 바람직하게는 0.1~50 중량%, 더 바람직하게는 0.5~25 중량%이나, 이에 한정되는 것은 아니다. The strain content of the present invention, which is an active ingredient in the food composition according to the present invention, is 0.01 to 99% by weight, preferably 0.1 to 50% by weight, more preferably 0.5 to 25% by weight, based on the total weight of the composition. It is not limited.
또한, 본 발명의 균주를 식품 첨가제의 형태로 사용하기 위해서는 분말 또는 농축액 형태로 제조하여 사용할 수 있다.In addition, in order to use the strain of the present invention in the form of a food additive, it may be prepared and used in the form of a powder or a concentrate.
상기 본원 발명의 약학적 조성물 및 식품 조성물에 있어서, 유효 성분의 혼합량은 그의 사용 목적(예방, 건강 또는 증상완화)에 따라 적합하게 결정될 수 있으며, 이에 제한되지 않으나 예를 들어 본 발명에 따른 균주를 10 8CFU/g 내지 10 20CFU/g 농도로 포함할 수 있다. 본 발명의 균주의 유효용량은 상기 농도 범위에 준해서 사용할 수 있으나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며, 유효성분은 안전성 면에서 아무런 문제가 없기 때문에 상기 범위 이상의 양으로도 사용될 수 있다.In the pharmaceutical composition and food composition of the present invention, the mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or symptom relief), but is not limited thereto, for example, the strain according to the present invention 10 8 CFU/g to 10 20 CFU/g. The effective dose of the strain of the present invention may be used in accordance with the above concentration range, but in the case of long-term intake for health and hygiene purposes or for health control purposes, it may be less than the above range, and the active ingredient is safe Since there is no problem, it may be used in an amount exceeding the above range.
또한 본Also seen 발명은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 생균제 또는 정장용 조성물을 제공한다. The present invention provides a probiotic or dressing composition comprising the strain or a culture thereof as an active ingredient.
본 발명에 따른 신규한 균주는 인간 및 동물의 건강 증진을 위한 정장용 생균제 또는 동물을 위한 사료용 생균제 조성물로 사용될 수 있다. 상기 조성물은 상기 균주 자체, 또는 이의 배양물 등을 유효성분으로 포함하며, 부형제 또는 담체를 추가로 포함할 수 있다. 조성물 내 본 발명의 신규한 균주의 함량은 조성물의 용도 및 제형에 따라 달라질 수 있다. The novel strain according to the present invention may be used as a probiotic for probiotic formulations for the promotion of human and animal health or a probiotic composition for feed for animals. The composition includes the strain itself, or a culture thereof as an active ingredient, and may further include an excipient or a carrier. The content of the novel strain of the present invention in the composition may vary depending on the composition and use of the composition.
본 발명에 따른 정장용 또는 생균제 조성물은 다양한 제형과 방법으로 제조 및 투여될 수 있다. 예를 들어, 본 발명에 따른 신규한 균주 또는 이의 배양물을 약제학적 분야에서 통상적으로 사용하는 담체 및 향료와 혼합하여 정제(tablet), 트로키(troche), 캡슐(capsule), 엘릭실(elixir), 시럽(syrup), 산제(powder), 현탁제(suspension) 또는 과립제(granule) 등의 형태로 제조 및 투여될 수 있다. 담체로는 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제 등을 사용할 수 있다. Formal or probiotic compositions according to the present invention can be prepared and administered in a variety of formulations and methods. For example, a novel strain or a culture thereof according to the present invention is mixed with a carrier and a fragrance commonly used in the pharmaceutical field, and then tablets, troches, capsules, elixirs ), syrup, powder, suspension, or granules. As the carrier, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, etc. can be used.
투여 방식은 경구, 비경구 또는 도포법 등을 사용할 수 있다. 또한, 투여용량은 체내에서 활성성분의 흡수도, 불활성율 및 배설속도, 피투여자의 연령, 성별, 상태 등에 따라 적절히 선택할 수 있다. The administration method may be oral, parenteral or application. In addition, the dosage can be appropriately selected according to the absorbance of the active ingredient in the body, the rate of inactivation and excretion, the age, sex, and condition of the recipient.
또한 본 발명에 따른 신규한 균주 또는 이의 배양물은 김치, 음료, 이유식 등의 식품에 대한 식품첨가제로, 특히 발효제품 제조를 위한 스타터(starter)로 사용될 수 있다. 상기 '발효제품'은 본 명세서에서 용어 '발효식품'과 혼용되어 지칭될 수 있다. 상기 발효제품은 제조시 발효공정이 필요한 식품으로서 당업계에 알려진 것이라면 그 종류가 제한되지 않으나, 예를 들어, 치즈, 요거트, 버터, 크림, 아이스크림, 유산균 음료, 캐피어, 발효유, 김치, 발효생식제품 등을 포함한다. 본 발명의 신규한 균주를 이용한 발효제품은 당업계에 공지된 통상의 방법에 따라 제조될 수 있다. 발효제품의 제조방법은 통상적으로 원료의 준비, 유산균의 첨가, 발효, 완성된 제품의 회수 등의 과정으로 이루어진다. 원료의 준비 단계는 발효 대상이 되는 재료를 준비하고, 발효가 잘 이루어지도록 발효 조건을 준비하는 단계이다. 유산균의 첨가는 발효 대상의 양에 따라 적량의 균을 첨가하는 것이다. 발효는 통상의 발효균의 발효조건에 따라 수행되는 것으로 예를 들어 20℃ 내지 40℃에서 1 내지 168시간 동안 수행될 수 있다. 완성된 제품의 회수는 발효물에서 불필요한 부산물이나 미발효된 재료를 제거하는 것에서부터 저장, 운반을 용이하게 하기 위한 모든 후처리 과정 및 포장 등을 포함한다.In addition, the novel strains or cultures thereof according to the present invention can be used as food additives for foods such as kimchi, beverages, baby foods, and in particular, as starters for manufacturing fermented products. The'fermented product' may be referred to herein interchangeably with the term'fermented food'. The fermentation product is a food that requires a fermentation process at the time of manufacture, and if known in the art, its type is not limited, for example, cheese, yogurt, butter, cream, ice cream, lactic acid bacteria drink, capeer, fermented milk, kimchi, fermentation Products and the like. Fermentation products using the novel strain of the present invention can be prepared according to conventional methods known in the art. The method of manufacturing fermented products usually consists of the preparation of raw materials, the addition of lactic acid bacteria, fermentation, and recovery of the finished product. The preparation step of the raw material is a step of preparing the material to be fermented and preparing the fermentation conditions so that the fermentation is performed well. The addition of lactic acid bacteria is to add an appropriate amount of bacteria according to the amount of the fermentation target. Fermentation is performed according to the fermentation conditions of a conventional fermentation bacterium, and may be performed, for example, at 20°C to 40°C for 1 to 168 hours. Recovery of the finished product includes removing unnecessary by-products or unfermented materials from the fermentation product and all post-processing procedures and packaging to facilitate storage and transportation.
또한 본Also seen 발명은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 면역조절용 조성물을 제공한다. The present invention provides a composition for immunomodulation comprising the strain or a culture thereof as an active ingredient.
면역조절에 관여하는 "세포"는 인터류킨-2(IL-2), 인터페론 γ(IFN-γ), 및 TNF-α 등을 생산하는 Th1 세포와 IL-4, IL-5, IL-6, IL-10, 및 IL-13 등을 생산하는 Th2세포로 구성된다. 이들 Thl과 Th2세포가 생산하는 사이토카인에 의해 조절되는 면역균형을 Thl/Th2 균형이라고 하며, Thl세포는 세포성 면역의 조절에 중요하고, Th2세포는 체액성 면역의 조절에 중요한 역할을 한다고 알려져 있다. 정상상태에서는 Thl의 분화에 중요한 IFN-γ를 중심으로 한 사이토카인과 Th2의 분화에 중요한 IL-4를 주로한 사이토카인이 서로 간섭하면서 Thl/Th2 균형을 일정하게 유지하고 있다. 그러나 Thl/Th2 균형이 깨지면 다양한 면역질환을 유발할 수 있는데, Thl에 편향한 경우에는 세포성 면역이 부활하여 감염저항성이 증강하고, Th2에 편향한 경우에는 감염저항성이 감소하고, 역으로 알레르기가 증강한다.The "cells" involved in immunomodulation are Th1 cells producing interleukin-2 (IL-2), interferon γ (IFN-γ), and TNF-α, and IL-4, IL-5, IL-6, IL -10, and Th-13 cells that produce IL-13. Immune balance regulated by cytokines produced by these Thl and Th2 cells is called Thl/Th2 balance, Thl cells are important for the regulation of cellular immunity, and Th2 cells are known to play an important role in the regulation of humoral immunity. have. Under normal conditions, the cytokine centering on IFN-γ, which is important for the differentiation of Thl, and the cytokine mainly IL-4, which is important for the differentiation of Th2, interfere with each other, maintaining a constant Thl/Th2 balance. However, if the Thl/Th2 balance is broken, it can cause various immune diseases. In the case of bias to Thl, cellular immunity is revived to increase infection resistance, and when to Th2 bias, infection resistance decreases, and conversely, allergy increases. do.
또한 본 발명에 따른 효모는 Th2 사이토카인(예: IL-4, IL-5)을 억제하거나 또는/및 Th1 사이토카인(예: IL-12, IFN-γ, TNF-α)을 유도함으로써 Th1/Th2 면역반응의 균형(balance)을 조절하는 생리적 활성을 가진다. 구체적인 생리적 활성은 다음과 같다. 1) 오브알부민 감작 마우스에서 IL-12, IFN-γ, TNF-α의 생산을 증가시킨다; 2) 마우스 혈청에서 IgE 수준을 감소시킨다. 3) 호산구, 호중구, 호염구 비율을 감소시킨다. 4) Th2-림프구에 의해 매개되는 사이토카인(IL-4 및 IL-10)의 발현을 억제시킨다; 5) 아토피피부염 병변 부위에서 호산구 및 비만세포 침윤을 감소시킨다.In addition, the yeast according to the present invention inhibits Th2 cytokines (eg IL-4, IL-5) or/and induces Th1 cytokines (eg IL-12, IFN-γ, TNF-α) Th1/ It has a physiological activity that regulates the balance of the Th2 immune response. The specific physiological activity is as follows. 1) increase production of IL-12, IFN-γ, TNF-α in ovalbumin-sensitized mice; 2) Reduce IgE levels in mouse serum. 3) Reduce the ratio of eosinophils, neutrophils, and basophils. 4) inhibit the expression of cytokines (IL-4 and IL-10) mediated by Th2-lymphocytes; 5) Reduces eosinophil and mast cell infiltration at the site of atopic dermatitis lesions.
또한 본Also seen 발명은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 식품첨가용 조성물을 제공한다. The present invention provides a food additive composition comprising the strain or a culture thereof as an active ingredient.
본 발명에 따른 본 발명의 균주 또는 이의 배양물은 김치, 음료, 이유식, 발효유, 빵 등의 식품에 대한 식품첨가제로 사용될 수 있다. 아울러, 본 발명의 균주 또는 이의 배양물은 발효제품 제조를 위한 스타터(starter)로 사용될 수 있다. 상기 발효제품은 치즈, 김치, 발효생식제품 등을 포함한다. 본 발명의 균주 또는 이의 배양물을 이용한 발효제품은 당업계에 공지된 통상의 방법에 따라 제조될 수 있다.예컨대, 현미와 율무 등의 곡류 분말에 본 발명에 따른 균주 또는 이를 포함하는 2종 이상의 혼합 유산균을 처리하여 적정 온도에서 발효시킨 후, 백태, 찹쌀, 수수 등의 다양한 농산물을 영양적인 균형과 기호성이 우수하도록 적절히 배합하여 발효 생식제품을 제조할 수 있다.The strain of the present invention or cultures thereof according to the present invention can be used as a food additive for foods such as kimchi, beverages, baby food, fermented milk, and bread. In addition, the strain or culture of the present invention may be used as a starter for manufacturing fermented products. The fermented products include cheese, kimchi, fermented products, and the like. The fermentation product using the strain of the present invention or a culture thereof may be prepared according to a conventional method known in the art. For example, two or more strains comprising the strain according to the present invention or two or more thereof containing cereal grains such as brown rice and yulmu After processing the mixed lactic acid bacteria and fermenting at an appropriate temperature, fermented reproductive products can be prepared by appropriately blending various agricultural products such as baektae, glutinous rice, and sorghum to have excellent nutritional balance and palatability.
본 발명은 염증성 질환의 예방 또는 치료용 제제를 제조하기 위한 상기 균주 또는 이의 배양물의 용도를 제공한다.The present invention provides the use of the strain or a culture thereof for preparing an agent for the prevention or treatment of inflammatory diseases.
본 발명은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 조성물의 유효량을 이를 필요로 하는 개체에 투여하는 것을 포함하는 염증성 질환의 치료 방법을 제공한다.The present invention provides a method of treating an inflammatory disease comprising administering an effective amount of a composition comprising the strain or culture thereof as an active ingredient to an individual in need thereof.
본 발명은 생균제를 제조하기 위한 상기 균주 또는 이의 배양물의 용도를 제공한다.The present invention provides the use of the strain or its culture for producing a probiotic.
본 발명은 정장용 제제를 제조하기 위한 상기 균주 또는 이의 배양물의 용도를 제공한다.The present invention provides the use of the strain or its culture for preparing a formulation for dressing.
본 발명은 면역조절용 제제를 제조하기 위한 상기 균주 또는 이의 배양물의 용도를 제공한다.The present invention provides the use of the strain or its culture for preparing an agent for immunomodulation.
본 발명은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 조성물의 유효량을 이를 필요로 하는 개체에 투여하는 것을 포함하는 면역조절의 방법을 제공한다.The present invention provides a method of immunomodulation comprising administering an effective amount of a composition comprising the strain or culture thereof as an active ingredient to an individual in need thereof.
본 발명은 식품첨가용 제제를 제조하기 위한 상기 균주 또는 이의 배양물의 용도를 제공한다.The present invention provides the use of the strain or its culture for preparing a food additive preparation.
본 발명의 상기 ‘유효량’이란 개체에게 투여하였을 때, 염증성 질환의 개선, 치료, 예방, 검출, 진단 또는 염증성 질환의 억제 또는 감소 효과를 나타내는 양을 말하며, 상기 ‘개체’란 동물, 바람직하게는 포유동물, 특히 인간을 포함하는 동물일 수 있으며, 동물에서 유래한 세포, 조직, 기관 등일 수도 있다. 상기 개체는 상기 효과가 필요한 환자(patient) 일 수 있다.The'effective amount' of the present invention refers to an amount that, when administered to an individual, shows an effect of improving, treating, preventing, detecting, diagnosing or suppressing or reducing an inflammatory disease, and the'individual' is an animal, preferably It may be an animal including mammals, particularly humans, or cells, tissues, organs, etc. derived from animals. The subject may be a patient in need of the effect.
본 발명의 상기 ‘치료’는 염증성 질환 또는 염증성 질환의 증상을 개선시키는 것을 포괄적으로 지칭하고, 이는 상기 질환을 치유하거나, 실질적으로 예방하거나, 또는 상태를 개선시키는 것을 포함할 수 있으며, 상기 질환으로부터 비롯된 한 가지 증상 또는 대부분의 증상을 완화시키거나, 치유하거나 예방하는 것을 포함하나, 이에 제한되는 것은 아니다.The'treatment' of the present invention broadly refers to improving the symptoms of an inflammatory disease or inflammatory disease, which may include curing, substantially preventing, or improving the condition, from the disease It includes, but is not limited to, alleviating, healing or preventing one symptom or most of the symptoms.
본 발명에 따른 신규한 효모는 Treg이 매개하는 Th2 면역반응을 촉진하여 항염증 활성을 가지며, 정장 효과를 가지고, 오브알부민 감작 마우스 모델에 대하여 피부 및 장에서 염증을 억제하는 효과가 탁월하다. 뿐만 아니라, 내산성 및 내담즙성 또한 우수하여 상업적으로 이용가치가 높다. The novel yeast according to the present invention promotes a Th2 immune response mediated by Treg, has anti-inflammatory activity, has a formal effect, and has an excellent effect of suppressing inflammation in the skin and intestine against the ovalbumin-sensitized mouse model. In addition, it has excellent acid resistance and bile resistance, and thus has high commercial value.
도 1은 실험을 위한 OVA 감작 쥐에서의 아토피 피부염 유도 실험 설계와 계획표를 나타낸 그림이다.1 is a diagram showing a design and a schedule of atopic dermatitis induction experiment in OVA-sensitized mice for the experiment.
도 2는 카자츠타니아 투리센시스 CAU Y1706의 주사전자현미경(SEM) 사진이다.FIG. 2 is a scanning electron microscope (SEM) photograph of Kauriztania Turisensis CAU Y1706.
도 3a 내지 3g는 카자츠타니아 투리센시스 CAU Y1706 의 경구투여가 사이토카인 농도에 미치는 영향을 확인한 결과이다. 수치를 분석하는 데에 혈청을 사용하였으며, ELIZA kit를 이용하였다. 유의 표시는 양성그룹의 ANOVA 분석값과 평균값의 차이를 나타낸다. *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001.3A to 3G are results confirming the effect of oral administration of Kazaztania Turisensis CAU Y1706 on cytokine concentration. Serum was used to analyze the values, and an ELIZA kit was used. Signs of significance indicate the difference between the ANOVA analysis value of the positive group and the mean value. *p <0.05; **p <0.005; ***p <0.0005; ****p <0.0001.
도 3a는 IL-4 농도에 미치는 영향을 확인한 결과이다.Figure 3a is a result confirming the effect on the IL-4 concentration.
도 3b는 IL-5 농도에 미치는 영향을 확인한 결과이다.Figure 3b is a result confirming the effect on the IL-5 concentration.
도 3c는 IL-10 농도에 미치는 영향을 확인한 결과이다.Figure 3c is a result confirming the effect on the IL-10 concentration.
도 3d는 IL-12 농도에 미치는 영향을 확인한 결과이다.Figure 3d is a result confirming the effect on the IL-12 concentration.
도 3e는 IFN-γ 농도에 미치는 영향을 확인한 결과이다.Figure 3e is a result confirming the effect on the IFN-γ concentration.
도 3f는 TNF-α 농도에 미치는 영향을 확인한 결과이다.Figure 3f is a result confirming the effect on the TNF-α concentration.
도 3g는 IL-1β 농도에 미치는 영향을 확인한 결과이다.Figure 3g is a result confirming the effect on the concentration of IL-1β.
도 4a 내지 4e는 카자츠타니아 투리센시스 CAU Y1706의 경구투여가 혈액에 미치는 영향을 확인한 결과이다. 염증은 전혈 분석으로 확인되었다. 유의 표시는 양성그룹의 ANOVA 분석값과 평균값의 차이를 나타낸다. * p < 0.05; ** p < 0.005; *** p < 0.0005; **** p < 0.0001.4A to 4E are results confirming the effect of oral administration of Kazaztania Turisensis CAU Y1706 on blood. Inflammation was confirmed by whole blood analysis. Signs of significance indicate the difference between the ANOVA analysis value of the positive group and the mean value. * p < 0.05; ** p <0.005; *** p <0.0005; **** p <0.0001.
도 4a는 알레르기 반응을 확인하기 위해 혈청 내 IgE 수치를 ELISA 분석으로 측정한 결과이다.Figure 4a is a result of measuring the IgE level in the serum by ELISA analysis to confirm the allergic reaction.
도 4b는 호산구 비율을 확인한 결과이다. Figure 4b is the result confirming the eosinophil ratio.
도 4c는 호산구 수를 확인한 결과이다.4c is a result of confirming the number of eosinophils.
도 4d는 호중구 비율을 확인한 결과이다.4D is a result of confirming the neutrophil ratio.
도 4e는 호염구 비율을 확인한 결과이다.Figure 4e is a result of checking the basophil ratio.
도 5a 내지 5h는 카자츠타니아 투리센시스 CAU Y1706의 경구투여가 아토피 피부염 유발 쥐의 장내미생물 집단의 풍부도와 다양도에 기여함을 확인한 결과이다.5A to 5H are results confirming that oral administration of Kazaztania thuricensis CAU Y1706 contributes to the abundance and diversity of the intestinal microbial population of atopic dermatitis-induced mice.
도 5a는 Shannon-wiener 지수를 측정한 결과이다(97% 유사성으로 정의).Figure 5a is a result of measuring the Shannon-wiener index (defined by 97% similarity).
도 5b는 Simpson 지수를 측정한 결과이다. 5B is a result of measuring the Simpson index.
도 5c는 Unweighted UniFrac 기반 (PCoA) 3D 도표로 장내미생물군의 차이를 추산한 결과이다(파란색 표지: 음성대조군; 빨간색 표지: 양성대조군; 초록색 표지: K. turicensis CAU Y1706 투여군; 보라색 표지: 베토타스틴베실산염 투여군).Figure 5c is a result of estimating the difference in the intestinal microflora in an Unweighted UniFrac based (PCoA) 3D chart (blue label: negative control; red label: positive control; green label: K. turicensis CAU Y1706 administered group; purple label: betota Stinbesylate group).
도 5d는 속 단위의 상대 존재비를 나타낸 결과이다. Figure 5d is a result showing the relative abundance of the genera unit.
도 5e는 Faecalibacterium 속의 산점도를 나타낸 결과이다.Figure 5e is a result showing the scatter plot of Faecalibacterium genus.
도 5f는 Akkermansia 속의 산점도를 나타낸 결과이다.Figure 5f is a result showing the scatter plot of the genus Akkermansia.
도 5g는 Lactobacillus 속의 산점도를 나타낸 결과이다.Figure 5g is a result showing the scatter plot in the genus Lactobacillus.
도 5h는 Ruminococcus 속의 산점도를 나타낸 결과이다.Figure 5h is a result showing the scatter plot in the genus Ruminococcus.
도 5e 내지 도 5h의 네 그룹 비교에서 비모수 Kruskal-Wallis test에 의해 통계학적 차이가 드러났다 ( p < 0.05).Statistical differences were revealed by the nonparametric Kruskal-Wallis test in the comparison of the four groups of FIGS. 5E-5H ( p < 0.05).
도 6a 내지 6d는 카자츠타니아 투리센시스 CAU Y1706의 경구투여가 쥐의 아토피 피부염 피부 병변에 미치는 영향을 확인한 결과이다. 유의 표시는 양성그룹의 ANOVA 분석값과 평균값의 차이를 나타낸다. *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001.6A to 6D are results confirming the effect of oral administration of Kazaztania Turisensis CAU Y1706 on atopic dermatitis skin lesions in rats. Signs of significance indicate the difference between the ANOVA analysis and the mean value of the positive group. *p <0.05; **p <0.005; ***p <0.0005; ****p <0.0001.
도 6a는 마우스 등 피부의 아토피 병변을 피부염 지수로 평가하였으며 피부염 점수는 홍반, 건조함, 쥐가 긁은 정도의 세 가지 증상의 점수 합으로 계산한 것이다.FIG. 6A shows a dermatitis index for atopic lesions of skin, such as mice, and dermatitis scores are calculated as the sum of the scores of the three symptoms of erythema, dryness, and scratching of mice.
도 6b는 마우스 등 피부의 파라핀 블록을 톨루이딘블루로 염색하여 현미경을 이용하여 비만세포 수를 측정한 결과이다(축척막대=100 μm).Figure 6b is a result of measuring the number of mast cells using a microscope by staining the paraffin block of the skin, such as a mouse with toluidine blue (scale bar = 100 μm).
도 6c는 마우스 회장 부위의 파라핀 블록을 톨루이딘블루로 염색하여 현미경을 이용하여 비만세포의 수를 측정한 결과이다(축척막대 = 100 μm).Figure 6c is a result of measuring the number of mast cells using a microscope by staining the paraffin block of the mouse ileum with toluidine blue (scale bar = 100 μm).
도 6d는 마우스 등 피부조직을 콩고레드로 염색후 현미경 분석을 통하여 호산구 수를 정량한 결과이다.Figure 6d is a result of quantifying the number of eosinophils through microscopic analysis after staining skin tissues such as mice with Congo red.
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.However, the following examples are only to illustrate the present invention, the content of the present invention is not limited to the following examples.
실험재료 및 방법 Experimental materials and methods
KazachstaniaKazachstania turicensisturicensis CAUCAU Y1706의 분리 및 식별 Separation and identification of Y1706
김치 샘플들 대한민국 서울의 재래시장에서 입수되었다. 샘플은 모두 잘게 자른 뒤 10mL의 펩톤 용액 (0.85% mass/vol)과 혼합하였다. 단계적으로 희석한 후 샘플을 MRS (Man, Rogosa, and Sharpe; BD BBL, Spatks, MD. USA) 고체배지에 도말하고 30℃의 MIR-254-PK 배양기 (Panasonic, Osaka, Japan) 에서 2-3일간 배양하였다. 순수 분리를 위해 흰색의 콜로니를 선별하여 대두카제인소화한천배지 (Tryptic Soy Agar-TSA; BD BBL)에 몇 번의 분리배양을 거쳤으며, 모든 분리배양은 30℃에서 24시간 동안 이루어졌다. 효모균 분리와 식별을 위해 rRNA 유전자 (18S, 26S, 5.8-ITS) 서열 분석법을 사용하였으며, 3730 automatic DNA sequencer (Applied Biosystems, Foster City, CA, USA) 을 분석에 사용하였다. 유전자 서열은 NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) 을 통해 분석하였다. 선별된 효모는 30℃의 조건에서 TSA에 배양한 후, 광학현미경인 DM 1000 light (Leica, Wetzlar, Germany) 와 주사전자현미경인 SIGMA field-emission scanning electron microscope (Carl Zeiss, Dresden, Germany) 를 이용하여 세포 외관을 관찰하였다. 생화학적 특성은 API 20C AUX와 API 50CH을 사용하여 확인하였으며, 제조사 (biomerieux, Marcy-l' E Toile, France) 의 지침을 따라 수행되었다. 분리체 중 하나인 K. turicensis CAU Y1706이 아토피 피부염 연구에 사용되었다.Kimchi samples were obtained from a traditional market in Seoul, Korea. All samples were chopped and mixed with 10 mL of peptone solution (0.85% mass/vol). After the stepwise dilution, the samples are spread on MRS (Man, Rogosa, and Sharpe; BD BBL, Spatks, MD. USA) solid medium and 2-3 in a MIR-254-PK incubator at 30°C (Panasonic, Osaka, Japan). Cultured daily. For pure separation, white colonies were screened and subjected to several separation cultures on Tryptic Soy Agar-TSA (BD BBL), and all separation cultures were performed at 30° C. for 24 hours. For isolation and identification of yeast cells, rRNA gene (18S, 26S, 5.8-ITS) sequence analysis was used, and 3730 automatic DNA sequencer (Applied Biosystems, Foster City, CA, USA) was used for analysis. Gene sequences were analyzed via NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The screened yeast was cultured in TSA at a condition of 30° C., and then used an optical microscope DM 1000 light (Leica, Wetzlar, Germany) and a scanning electron microscope SIGMA field-emission scanning electron microscope (Carl Zeiss, Dresden, Germany). Cell appearance was observed. Biochemical properties were confirmed using API 20C AUX and API 50CH, and were performed according to the manufacturer's instructions (biomerieux, Marcy-l' E Toile, France). One isolate, K. turicensis CAU Y1706, was used in the study of atopic dermatitis.
이 같이 선별된 카자츠타니아 투리센시스 균주를 CAU Y1706로 명명하였다. 분리동정된 신규한 카자츠타니아 투리센시스 균주는 한국생명공학연구원 생물자원센터(KCTC)에 2019년 01월 22일 기탁하였다(수탁번호 : KCTC13794BP).The thus-selected Kazaztania thuricensis strain was named CAU Y1706. The newly identified strains of Kazaztania thuricensis were deposited with the Korea Research Institute of Bioscience and Biotechnology Biotechnology Center (KCTC) on January 22, 2019 (Accession No.: KCTC13794BP).
윤리 및 실험 설계Ethics and experimental design
동물실험은 한국 식품의약품안전관리본부의 지침에 따라 시행되었다. 쥐의 혈액샘플과 조직샘플은 중앙대학교 동물생명윤리위원회의 지침에 따라 채취되었다. 생후 5주차인 암컷 BALB/c 쥐 (40마리) 는 중앙실험동물 (서울, 대한민국) 에서 분양되었다. 실험 설계도와 계획표는 도 1과 같다. 모든 쥐는 음성대조군 (negative control, OVA 비감작 + PBS), 양성대조군 (positivw control, OVA 감작 + PBS), K. turicensis CAU Y1706군 (OVA 감작 + K. turicensis CAU Y1706), 베포타스틴베실산염군 (OVA 감작 + 베포타스틴베실산염)의 네 그룹에 각 10마리씩 무작위적으로 배치되었다. 피부감작을 통한 아토피 피부염 유도를 위해 모든 쥐들을 이발기와 제모크림으로 제모하였다. OVA grade V (Sigma-Aldrich, St. Louis, MI, USA) (50 mg/mL)와 명반 (Sigma-Aldrich)을 인산완충생리식염수 (PBS) 에 용해시킨 수용액을 실험 시작일로부터 7일, 21일, 35일, 49일 째에 복강 내 주사하였으며, 절차에 따라 8주간 피부 위에 직접 감작하였다. 동결건조 된 K. turicensis CAU Y1706 (1 ×10 10CFU/mouse) 는 200 μl의 인산완충생리식염수에 용해시킨 후 경구투여 하였다. 음성대조군은 8주간 같은 용량의 인산완충생리식염수를 투여하였다. 양성처리대조군의 베포타스틴베실산염 (Sigma-Aldrich)은 하루에 한 번 경구투여 하였다 (0.5 mg/kg). 8주 후, 분석을 위해 쥐들을 희생하였으며, 등 피부와 회장, 혈액을 채취하였다.Animal experiments were conducted in accordance with the guidelines of the Korea Food and Drug Safety Management Headquarters. Rat blood samples and tissue samples were collected according to the guidelines of Chung-Ang University Animal Bioethics Committee. Female BALB/c rats (40 animals), 5 weeks old, were pre-sold in central laboratory animals (Seoul, Korea). The experimental design and planning table are shown in FIG. 1 . All mice were negative control (negative control, no OVA + PBS), positive control (positivw control, OVA sensitization + PBS), K. turicensis CAU Y1706 group (OVA sensitization + K. turicensis CAU Y1706), bepotastine besylate group (OVA sensitization + bepotastine besylate) were randomly assigned to each of 10 animals in four groups. To induce atopic dermatitis through skin sensitization, all rats were epilated with a barber and epilation cream. An aqueous solution of OVA grade V (Sigma-Aldrich, St. Louis, MI, USA) (50 mg/mL) and alum (Sigma-Aldrich) dissolved in phosphate buffered physiological saline (PBS) 7 days, 21 days from the start of the experiment , Injected on the 35th and 49th day intraperitoneally, and sensitized directly onto the skin for 8 weeks according to the procedure. Lyophilized K. turicensis CAU Y1706 (1 × 10 10 CFU/mouse) was orally administered after dissolving in 200 μl of phosphate buffered saline. The negative control group received the same dose of phosphate buffered saline for 8 weeks. Bepotastine besylate (Sigma-Aldrich) of the positive control group was administered orally once a day (0.5 mg/kg). Eight weeks later, rats were sacrificed for analysis, and back skin, ileum and blood were collected.
혈액 및 혈청 사이토카인 분석Blood and serum cytokine analysis
혈액샘플은 캐필러리를 이용한 안와채혈을 통해 수득되었다. 호산구 분석과 수 확인을 위한 전혈은 EDTA 채혈관 (Green Cross Laboratories, 용인, 대한민국) 을 이용하여 채취되었다. IgE와 사이토카인(IL-4, IL-5, IL-10, IL-12, IL-1β, TNF-α, IFN-γ) 분석을 위한 혈청은 4℃에서 1시간동안 응고시킨 후 1시간의 5,000 ×g 원심분리 과정을 거쳐 혈전을 가라앉힌 후 수득하였다. 사이토카인은 ELISA kit를 이용하여 확인하였으며, 제조사 (R&D systems, Minneapolis, MN. USA) 의 지침을 따라 수행하였다. 450nm에서의 흡광도를 확인하기 위해 an Infinite　200 PRO NanoQuant microplate reader (Tecan, Mannedorf, Switzerland) 를 이용하였다.Blood samples were obtained through orbital blood collection using capillaries. Whole blood for eosinophil analysis and number determination was collected using EDTA blood collection tubes (Green Cross Laboratories, Yongin, Korea). Serum for IgE and cytokine (IL-4, IL-5, IL-10, IL-12, IL-1β, TNF-α, IFN-γ) analysis was coagulated at 4° C. for 1 hour, followed by 1 hour. It was obtained after thriving the thrombus through a 5,000×g centrifugation process. Cytokines were identified using an ELISA kit, and were performed according to the manufacturer's instructions (R&D systems, Minneapolis, MN. USA). An Infinite 흡200 PRO NanoQuant microplate reader (Tecan, Mannedorf, Switzerland) was used to check the absorbance at 450 nm.
장내미생물 분석Intestinal microbial analysis
생후 16주의 쥐에서 채취한 배설물 샘플은 멸균된 2 mL의 튜브에 담아 즉시 얼음에 보관하였으며, 이후 -80℃에 보관하였다. DNA는 FastDNA SPIN kit for bacterial DNA를 이용하여 추출하였으며, 제조사 (MP Biomedicals, Santa Ana, CA, USA) 의 지침에 따라 수행되었다. 16S rRNA 유전자의 V3-V4 부위를 PCR을 통하여 증폭하였으며, MiSeq-based high-throughput sequencing (Illumina, San Diego, CA, USA) 으로 분석하였다. 염기서열화 후, 어댑터 서열을 the Scythe (v 0.994; https://github.com/vsbuffalo/scythe) 와 Sickle programs (https://github.com/vsbuffalo/scythe) 을 통해 제거하였다. 염기서열에서 너무 짧은 서열 (< 36 bp), extra-long tails, chimeric reads, 노이즈 서열은 CD-HIT-OTU (http://weizhong-lab.ucsd.edu/cd-hit-otu) 을 통해 잘라내었다 (Li et al., 2012). 최소한의 quality scores와 길이는 각각 >20, 300 bp 로 제한하였다. 나머지 대표 서열은 조합하였으며 operational taxonomic units는 CD-HIT-OTU를 통하여 97%의 유사도로 정의하였다. 장내미생물 분석은 QIIME (Quantitative Insight Into Microbial Ecology; v.1.9.1) 소프트웨어를 사용하여 이루어졌다.Excrement samples collected from rats at 16 weeks of age were immediately stored in ice in a sterile 2 mL tube, and then stored at -80°C. DNA was extracted using FastDNA SPIN kit for bacterial DNA, and was performed according to the manufacturer's instructions (MP Biomedicals, Santa Ana, CA, USA). The V3-V4 region of the 16S rRNA gene was amplified through PCR and analyzed by MiSeq-based high-throughput sequencing (Illumina, San Diego, CA, USA). After sequencing, the adapter sequence was removed through the Scythe (v 0.994; https://github.com/vsbuffalo/scythe) and Sickle programs (https://github.com/vsbuffalo/scythe). Sequences that are too short in sequence (< 36 bp), extra-long tails, chimeric reads, and noise sequences are cut through CD-HIT-OTU (http://weizhong-lab.ucsd.edu/cd-hit-otu) (Li et al., 2012). The minimum quality scores and length were limited to >20 and 300 bp respectively. The remaining representative sequences were combined and operational taxonomic units were defined with a similarity of 97% through CD-HIT-OTU. Intestinal microbial analysis was performed using QIIME (Quantitative Insight Into Microbial Ecology; v.1.9.1) software.
조직 분석Organizational analysis
희생된 쥐의 등 부위의 피부조직과 회장을 조직 분석에 사용하였다. 조직은 인산완충생리식염수를 이용해 10%로 희석한 포르말린 용액에 고정한 후 파라핀에 포매하였다. 조직 샘플은 마이크로톰을 이용하여 4-5 μm 두께로 자른 후 비만세포와 호산구 측정을 위해 각각 헤마톡실린과 에오신, 톨루이딘블루와 콩고레드로 염색하였다. 비만세포 수 측정을 위한 회장 조직 샘플의 모든 톨루이딘블루 염색 부위는 DM 4000B microscopy로 400배 배율에서 관찰하였다. 비만세포의 정량화를 위해 등 부위의 피부조직의 톨루이딘블루 염색부위 중 5개를 무작위로 선택하였으며, 호산구의 정량화를 위해 콩고레드 염색부위 중 20개를 무작위로 선택하였다.The skin tissue and ileum of the victim's back were used for tissue analysis. The tissue was fixed in a formalin solution diluted to 10% with phosphate buffered saline, and then embedded in paraffin. Tissue samples were cut using a microtome to a thickness of 4-5 μm, and stained with hematoxylin and eosin, toluidine blue and congo red for mast cells and eosinophil measurements, respectively. All toluidine blue staining sites of the ileum tissue samples for mast cell count were observed at 400x magnification by DM 4000B microscopy. For quantification of mast cells, 5 of the toluidine blue staining regions of the skin tissue of the back were randomly selected, and 20 of the congo red staining regions were randomly selected for quantification of eosinophils.
통계statistics
통계 분석에는 GraphPad Prism (v.7.0) software를 이용하였으며 데이터는 평균±표준오차로 표시하였다. 미생물의 강과 속의 상대적 차이를 식별하기 위한 그룹간의 비교는 비모수의 Kruskal-Wallis test에 의한 통계적 유의한 차이에 기초하였다. 사이토카인, 혈액샘플, 조직 분석에서의 유의미한 차이는 두 개 이상의 그룹을 비교하기 위한 ANOVA 분석으로 계산하였으며, p 값이 0.05 미만의 경우 유의미한 값으로 간주하였다.GraphPad Prism (v.7.0) software was used for statistical analysis, and data were expressed as mean±standard error. Comparisons between groups to identify the relative differences between microbial rivers and genus were based on statistically significant differences by the nonparametric Kruskal-Wallis test. Significant differences in cytokine, blood sample, and tissue analysis were calculated by ANOVA analysis to compare two or more groups, and were considered to be significant when the p value was less than 0.05.
실시예Example 1 : One : KazachstaniaKazachstania turicensisturicensis CAUCAU Y1706 의Y1706 분리 detach
K. turicensis CAU Y1706는 30℃의 환경에서 TSA 배지 (Bacto) 에 하루 간 배양 시 원형의 광택이 나는 크림색 콜로니를 형성하였다. 세포는 타원형이며 지름이 약 1.6-2.3μm 길이는 1.6-2.3μm로 관찰되었다 (도 2). K. turicensis CAU Y1706는 탄소공급원으로 D-glucose, D-galactose, D-saccharose, D-trehalose, D-raffinose, D-fructose, D-mannose, 2-keto-D-gluconate를 활용할 수 있는 것이 확인되었다. 반면 glycerol, L-arabinose, D-xylose, adonitol, xylitol, inositol, D-sorbitol, alpha-methyl-D-glucoside, N-acetyl-D-glucosamine, D-cellobiose, D-lactose, D-maltose, D-melezitose, D-ribose, D-mannitol, D-turanose, D-lyxose 는 탄소공급원으로 활용하지 못하는 것으로 나타났다. 100μl씩 처리하여 1시간 동안 반응시킨 후, 450nm에서 흡광도를 측정하였다.K. turicensis CAU Y1706 formed a round, shiny creamy colony when cultured in TSA medium (Bacto) for one day in an environment of 30°C. The cells were oval and the diameter was observed to be 1.6-2.3 μm and the length was 1.6-2.3 μm (FIG. 2 ). K. turicensis CAU Y1706 was confirmed to be able to utilize D-glucose, D-galactose, D-saccharose, D-trehalose, D-raffinose, D-fructose, D-mannose, 2-keto-D-gluconate as a carbon source. Became. Whereas glycerol, L-arabinose, D-xylose, adonitol, xylitol, inositol, D-sorbitol, alpha-methyl-D-glucoside, N-acetyl-D-glucosamine, D-cellobiose, D-lactose, D-maltose, D It was found that -melezitose, D-ribose, D-mannitol, D-turanose, and D-lyxose could not be used as carbon sources. After reacting for 100 hours by treating 100 μl, absorbance was measured at 450 nm.
실시예Example 2 : 사이토카인 수치에 대한 2: About cytokine levels KazachstaniaKazachstania turicensisturicensis CAUCAU Y1706의 효과 Effects of Y1706
K. turicensis CAU Y1706이 혈청 사이토카인 생성에 미치는 영향을 검사하였다. 양성대조군과 비교하였을 때, K. turicensis CAU Y1706 투여군의 혈청 내의 IL-4, IL-5와 같은 Th2 사이토카인 농도는 유의미하게 낮은 수치를 나타냈다. 베포타스틴베실산염 투여군 역시 낮은 수치를 나타냈다 (도 3a, 3b). 양성대조군과 비교 시, 혈청 내의 IL-10, IL-1β와 같은 Treg 사이토카인 수치 역시 K. turicensis CAU Y1706 투여군에서 더 낮게 나타났다 (도 3c, 3g). The effect of K. turicensis CAU Y1706 on serum cytokine production was examined. Compared to the positive control group, the concentrations of Th2 cytokines such as IL-4 and IL-5 in the serum of the K. turicensis CAU Y1706 administration group were significantly lower. The group administered with bepotastine besylate also showed low levels (FIGS. 3A and 3B ). Compared to the positive control group, Treg cytokine levels such as IL-10 and IL-1β in the serum were also lower in the K. turicensis CAU Y1706 administration group (FIGS. 3C and 3G ).
한편, IFN-γ, TNF-α, IL-12 와 같은 Th1 사이토카인의 수치는 양성대조군에 비해 K. turicensis CAU Y1706 투여군에서 더 높은 것이 확인되었다 (도 3d-3f). 이러한 결과는 K. turicensis CAU Y1706이 Treg이 매개하는 Th2 면역반응을 촉진하여 염증성 질환에 치료 효과가 뒷받침한다.On the other hand, it was confirmed that the levels of Th1 cytokines such as IFN-γ, TNF-α, and IL-12 were higher in the K. turicensis CAU Y1706 administration group than in the positive control group (FIGS. 3D-3F ). These results suggest that K. turicensis CAU Y1706 promotes Treg-mediated Th2 immune response, supporting the therapeutic effect on inflammatory diseases.
뿐만 아니라, 본 발명의 균주를 처리한 군이 대조군에 비하여 IFN-γ, TNF-α, IL-12 생산량이 월등히 우수한 것을 확인하였다.In addition, it was confirmed that the group treated with the strain of the present invention has superior IFN-γ, TNF-α, IL-12 production compared to the control group.
실시예Example 3 : 혈액, 혈청의 3: blood and serum IgEIgE 수치에 대한 For shame KazachstaniaKazachstania turicensisturicensis CAUCAU Y1706의 효과 Effects of Y1706
혈청 내 IgE 수치는 양성대조군에서 높게 나타났으며, K. turicensis CAU Y1706 투여군과 베포타스틴베실산염 투여군 에서는 눈에 띄게 낮아진 수치를 보였다 (도 4a). 뿐만 아니라 양성대조군, 베포타스틴베실산염 투여군과 K. turicensis CAU Y1706 투여군을 비교하였을 때, K. turicensis CAU Y1706 투여군의 호산구 수와 호산구, 호중구, 호염구의 비율이 눈에 띄게 감소한 것을 확인할 수 있었다 (도 4b-4e).Serum IgE levels were higher in the positive control group and K. turicensis In the CAU Y1706-administered group and the bepotastine besilate-administered group, there was a noticeably lowered value (FIG. 4A). In addition, the positive control, bepotastine besylate group and K. turicensis When comparing the CAU Y1706 administration group, K. turicensis It was confirmed that the ratio of eosinophils, eosinophils, neutrophils, and basophils in the CAU Y1706 administration group was remarkably decreased (FIGS. 4B-4E ).
실시예Example 4 : 4 : 장내미생물군의Intestinal microflora 조절에 대한 For adjustment KazachstaniaKazachstania turicensisturicensis CAUCAU Y1706의 효과 Effects of Y1706
장내미생물군의 다양성을 확인하기 위하여 배설물 샘플로부터 박테리아의 DNA를 추출하고, PCR을 통하여 16S rRNA 유전자 염기서열을 증폭하였다. 평균적으로 599 bp (±9.2 bp) 길이의 박테리아 염기서열을 샘플 1종 당 평균 80.778개, 총 3,231,137개를 수득하였다. 박테리아 집단의 다양성과 풍부도는 operational taxonomic units (OTUs) (97%로 정의) 와 Shannon and Simpson 지수를 통해 통계처리 되었다 (도 5a, 5b). 양성대조군에 비하여 K. turicensis CAU Y1706 투여군에서 장내미생물의 풍부도, 균등성, 다양성이 월등이 높은 것으로 나타났다. 속 수준에서의 장내미생물군의 프로파일 차이는 unweighted UniFrac 기반 3D PCoA를 통해 평가하였다 (도 5c). 이 결과들은 K. turicensis CAU Y1706 투여군의 장내미생물군이 양성대조군과 비교했을 때 유의미한 차이를 가진다는 것을 보여준다.To confirm the diversity of the intestinal microflora, bacterial DNA was extracted from the fecal sample, and the 16S rRNA gene sequence was amplified through PCR. On average, an average of 599 bp (±9.2 bp) of bacterial sequence was 80.778 per sample, and a total of 3,231,137 were obtained. The diversity and abundance of bacterial populations were statistically analyzed through operational taxonomic units (OTUs) (defined as 97%) and Shannon and Simpson indices (FIGS. 5A and 5B ). Compared with the positive control group, the K. turicensis CAU Y1706 administration group showed a superiority in intestinal microbial abundance, uniformity, and diversity. Differences in the profile of the gut microbiota at the genus level were evaluated through unweighted UniFrac based 3D PCoA (FIG. 5C ). These results show that the intestinal microbial group of the K. turicensis CAU Y1706 administration group had a significant difference compared to the positive control group.
문 단위에서 분석했을 때 총 11개의 박테리아 문이 발견되었으며, 가장 우세한 세 종류는 Firmicutes, Bacteroidetes, Verrucomicrobia 였다 (도 5d). When analyzed on a door-by-door basis, a total of 11 bacterial doors were found, and the three most prevalent were Firmicutes, Bacteroidetes, and Verrucomicrobia (FIG. 5D).
세 종류의 문에서 총 94개 속이 발견되었으며, 그 중 네 종류는 모든 실험 그룹에서 풍부히 발견되었다 (도 5e-5h). A total of 94 genera were found in the three types of gates, four of which were found abundantly in all experimental groups (FIGS. 5E-5H ).
Firmicutes 속에 포함되는 Faecalibacterium 은 양성대조군에 비하여 음성대조군과 K. turicensis CAU Y1706 투여군에서 수치가 낮은 것으로 나타났다 (p < 0.0001). 반대로 Ruminococcus 는 양성대조군에 비하여 음성대조군과 K. turicensis CAU Y1706 투여군에서 높은 수치를 보였다 (p < 0.05). Faecalibacterium contained in Firmicutes was found to be lower in the negative and K. turicensis CAU Y1706 groups compared to the positive control group (p <0.0001). Conversely, Ruminococcus showed higher values in the negative and K. turicensis CAU Y1706 groups compared to the positive control group (p <0.05).
Verrucomicrobia 문의 Akkermansia 는 양성대조군에 비하여 음성대조군과 K. turicensis CAU Y1706 투여군에서 풍부하였다 (p < 0.0001). 이 결과들은 양성대조군과 K. turicensis CAU Y1706 투여군에서 속 분류단위의 풍부도를 포함한 장내미생물군의 차이가 있음을 보여준다.Akkermansia of Verrucomicrobia was more abundant in the negative and K. turicensis CAU Y1706 groups compared to the positive control (p <0.0001). These results show that there is a difference in the intestinal microbiota including the abundance of the genus classification units in the positive control group and the K. turicensis CAU Y1706 administration group.
장내미생물군은 짧은사슬지방산 (Short chain fatty acids - SCFAs)을 생산하는 것으로 알려져있다. SCFA 부티라트는 선천적 면역력에 중요한 역할을 하며, 장 건강 보호, 항염증 효과, Treg세포 분화 촉진 등의 기능을 가진다. 또한 Bacteroides , Ruminococcus , Akkermansia는 부티라트를 생산하는 균으로, Lactobacillus 는 부티라트 생성 촉진과 관련이 있는 것으로 알려져 있다.The intestinal microbiota is known to produce short chain fatty acids (SCFAs). SCFA butyrat plays an important role in innate immunity and has functions such as protecting intestinal health, anti-inflammatory effects, and promoting Treg cell differentiation. Also Bacteroides , Ruminococcus , and Akkermansia are known to produce butyrat , and Lactobacillus is known to be involved in promoting butyrat production.
따라서, 본 발명의 효모를 경구투여한 결과 양성대조군과 비교하여 부티라트를 생성하거나 생성을 촉진하는 장내 미생물군이 다양하고 풍부하게 발견되었는 바, 본 발명의 효모의 정장제로 유용하게 사용할 수 있음을 확인하였다.Therefore, as a result of oral administration of the yeast of the present invention, a variety of intestinal microbial groups that produce or promote the production of butyrat compared to the positive control group have been found to be various and abundant. Confirmed.
실시예Example 5 : 피부 염증과 장에서의 5: skin inflammation and intestines KazachstaniaKazachstania turicensisturicensis CAUCAU Y1706의 영향 Impact of Y1706
K. turicensis CAU Y1706의 염증에 대한 효능을 검증하기 위해서, K. turicensis CAU Y1706를 투여한 OVA-감작 쥐의 피부염 점수를 이용한 평가와 병리학적 분석을 수행하였다. To verify the efficacy of K. turicensis CAU Y1706 on inflammation, evaluation and pathological analysis using dermatitis scores of OVA-sensitized rats administered K. turicensis CAU Y1706 were performed.
양성대조군에 비하여 K. turicensis CAU Y1706 투여군의 피부염 점수가 극적으로 낮은 것이 관찰되었다 (도 6a). 이 결과는 K. turicensis CAU Y1706이 아토피 피부염 쥐의 피부 병변 개선에 효과가 있다는 것을 나타낸다. It was observed that the dermatitis score of the K. turicensis CAU Y1706 administration group was dramatically lower than that of the positive control group (FIG. 6A ). These results indicate that K. turicensis CAU Y1706 is effective in improving skin lesions in atopic dermatitis rats.
또한, 등 피부세포와 회장 병변의 비만세포 국소침윤은 톨루이딘블루 염색을 통하여 확인하였다. K. turicensis CAU Y1706을 투여한 OVA-감작 쥐에서는 두 병변부위의 비만세포 축적이 억제된 것이 관찰되었다 (도 6b, 6c). In addition, local infiltration of mast cells of back skin cells and ileal lesions was confirmed by toluidine blue staining. In OVA-sensitized rats administered K. turicensis CAU Y1706, mast cell accumulation in both lesions was observed to be inhibited (FIGS. 6B and 6C ).
또한 양성대조군과 비교하였을 때 K. turicensis CAU Y1706을 투여한 OVA-감작 쥐에서는 호산구의 수가 확연히 적은 것이 확인되었다. 콩고레드로 염색한 등 피부병변 조직 관찰시, 베포타스틴베실산염 투여군과 마찬가지로 K. turicensis CAU Y1706 투여군의 호산구 침윤 수준이 감소된 것이 관찰되었다 (도 6d). 본 결과들은 K. turicensis CAU Y1706이 아토피 피부염 쥐의 등 피부와 회장에서 비만세포와 호산구의 침윤을 감소시키는 효과를 가짐을 보인다.In addition, it was confirmed that the number of eosinophils was significantly lower in OVA-sensitized rats administered K. turicensis CAU Y1706 when compared to the positive control group. When observing skin lesion tissues, such as stained with Congo red, it was observed that the level of eosinophilic infiltration of the group treated with K. turicensis CAU Y1706 was reduced as in the group treated with bepotastine besylate (FIG. 6D ). These results show that K. turicensis CAU Y1706 has the effect of reducing the infiltration of mast cells and eosinophils in the back skin and ileum of atopic dermatitis rats.
실시예Example 6 : 6: KazachstaniaKazachstania turicensisturicensis CAUCAU Y1706의 Y1706 내산성Acid resistance 평가 evaluation
생체 외 프로바이오틱스 특성 확인 실험으로 사람의 소화기관을 안정적으로 많은 수의 균이 살아서 장내까지 도달하기 위해 가져야 할 위액과 담즙에 대한 내성을 가져야 하므로 내산성, 내담즙성 실험을 많이 사용하고 있다.As an in vitro probiotic characterization experiment, acid- and bile-resistance experiments are frequently used because it is necessary to have resistance to gastric juice and bile, which a large number of bacteria must stably reach to the intestines by stably living human digestive organs.
이에, Kazachstania turicensis CAU Y1706의 산에 대한 내성을 규명하기 위하여, pH 2.0으로 적정한 TSB 액체배지(Pancreatic digest of Casein 17g/L, Papaic digest of Soybean 3g/L, Dextrose 2.5g/L, Sodium Chloride 5g/L, Dipotassium Phosphate 2.5g/L)에 접종하여 배양한 후 생균수를 측정하였다. 보다 구체척으로, 상기 Kazachstania turicensis CAU Y1706 균주를 TSB 액체배지에 접종 후, 30°에서 12시간 동안 배양하고 균체를 원심분리를 통해 회수한 후에, 멸균수로 OD 600값을 1.0으로 조절하여 현탁액 30μl를 pH 2.0으로 적정한 TSB 액체배지에 접종하여 30℃ 조건에서 0-120분 동안 배양한 후, 생균수를 Nanodrop (NanoQuant infinite M200, TECAN)으로 A600 nm에서 측정하였다.Thus, Kazachstania To characterize the acid resistance of turicensis CAU Y1706, TSB liquid medium titrated with pH 2.0 (Pancreatic digest of Casein 17g/L, Papaic digest of Soybean 3g/L, Dextrose 2.5g/L, Sodium Chloride 5g/L, Dipotassium After inoculating with Phosphate 2.5g/L), the number of viable cells was measured. More specifically, the Kazachstania After inoculating the turicensis CAU Y1706 strain into TSB liquid medium, incubating for 12 hours at 30°, and recovering the cells by centrifugation, adjusting the OD 600 value to 1.0 with sterile water to adjust the suspension 30μl to pH 2.0 TSB liquid After inoculating the medium and incubating at 30°C for 0-120 minutes, the viable cell count was measured at A600 nm with Nanodrop (NanoQuant infinite M200, TECAN).
실험결과 표 1에서 보는 바와 같이, CAU Y1706는 pH 2.0(위액과 동일 조건)의 MRS 배지에서 성장하는 능력을 보였다. CAU Y1706은 시간이 지날수록 균수가 상당한 속도로 증가하였으며, 이로서 강산의 조건에서 저해되지 않음을 확인하였다. 따라서 본원 발명의 CAU Y1706이 우수한 내산성을 가짐을 확인하였다. As shown in Table 1 , CAU Y1706 showed the ability to grow in MRS medium at pH 2.0 (same condition as gastric juice). CAU Y1706 confirmed that over time, the number of bacteria increased at a significant rate, and thus it was not inhibited under strong acid conditions. Therefore, it was confirmed that CAU Y1706 of the present invention has excellent acid resistance.
TimeTime Cell/ml (10 6)Cell/ml (10 6 )
00 8.00±0.008.00±0.00
1515 12.43±0.1512.43±0.15
3030 16.40±0.2616.40±0.26
6060 20.07±0.8020.07±0.80
120120 29.73±0.1129.73±0.11
실시예Example 7 : 7: KazachstaniaKazachstania turicensisturicensis CAUCAU Y1706의 Y1706 내담즙성Bile resistance 평가 evaluation
Kazachstania turicensis CAU Y1706의 담즙산에 대한 내성 특성을 실험하기 위하여, 담즙산(ox-bile dry pure, Merck, Germany)을 0, 0.2 및 0.4%(w/v)씩 첨가한 TSB 액체배지에 균주를 접종하여 배양한 후 생균수를 측정하였다. 보다 구체적으로, 상기 Kazachstania turicensis CAU Y1706 균주를 MRS 액체배지에 접종 후, 30℃에서 12시간 동안 배양하고 균체를 원심분리를 통해 회수한 후에, 멸균수로 OD 600값을 1.0으로 조절하여 현탁액 30μl를 0, 0.2 및 0.4%의 담즙산이 포함된 TSB 액체배지에 균주를 접종하여 30℃에서 0-12시간 배양한 후에, 생균수를 Nanodrop (NanoQuant infinite M200, TECAN)으로 A600 nm에서 측정하였다. Kazachstania In order to test the resistance characteristics of turicensis CAU Y1706 to bile acids, strains were inoculated into TSB liquid medium containing 0, 0.2 and 0.4% (w/v) bile acids (ox-bile dry pure, Merck, Germany) and cultured. After that, the viable cell count was measured. More specifically, the Kazachstania After inoculating the turicensis CAU Y1706 strain into the MRS liquid medium, incubating at 30° C. for 12 hours, and recovering the cells through centrifugation, adjusting the OD 600 value to 1.0 with sterile water to adjust the suspension 30 μl to 0, 0.2 and 0.4% After inoculation of the strain in TSB liquid medium containing bile acid, and cultured for 0-12 hours at 30°C, the viable cell count was measured at A600 nm with Nanodrop (NanoQuant infinite M200, TECAN).
실험결과 표 2 에서 보는 바와 같이, Kazachstania turicensis CAU Y1706는 0.2% 또는 0.4%의 담즙이 첨가된 TSB 배지에서 성장하는 능력을 보였으며, 0.2%와 0.4% 담즙에서 균이 우수한 성장 능력을 보였다. CAU Y1706은 담즙을 첨가하지 않은 대조군과 비교할 때, 1.25(0.2% 담즙 조건), 1.05(0.4% 담즙조건)의 높은 담즙저항(BTR)능을 가지는 것으로 확인되었다. 따라서, 상기 시험을 통하여 본 발명의 균주가 소화기관을 안정적으로 거쳐 많은 수의 균이 살아서 장내까지 도달할 수 있음을 확인하였다.As shown in Table 2 of the experimental results, Kazachstania turicensis CAU Y1706 showed the ability to grow in TSB medium to which 0.2% or 0.4% of bile was added, and the bacteria showed excellent growth ability in 0.2% and 0.4% of bile. CAU Y1706 was found to have a high bile resistance (BTR) ability of 1.25 (0.2% bile condition) and 1.05 (0.4% bile condition) compared to the control group without addition of bile. Therefore, it was confirmed through the test that the strain of the present invention stably passes through the digestive system and a large number of bacteria can survive and reach the intestine.
Cell counts as log incubation time (h) of Kazachstania turicensis CAU Y1706Cell counts as log incubation time (h) of Kazachstania turicensis CAU Y1706
Bile concentration (%)Bile concentration (%) 3h3h 6h6h 9h9h 12h12h BTRBTR (Bile Tolerance Rate)(Bile Tolerance Rate)
00 4.13±0.144.13±0.14 25.77±0.0625.77±0.06 44.60±0.0044.60±0.00 45.15±0.0545.15±0.05 1One
0.20.2 7.27±0.087.27±0.08 25.74±0.0625.74±0.06 46.13±1.5246.13±1.52 55.23±0.0655.23±0.06 1.251.25
0.40.4 5.57±0.055.57±0.05 17.26±0.4217.26±0.42 46.20±0.5246.20±0.52 52.47±0.0652.47±0.06 1.051.05
[수탁번호][Accession Number]
기탁기관명 : 한국생명공학연구원Depository name: Korea Research Institute of Bioscience and Biotechnology
수탁번호 : KCTC13794BPAccession number: KCTC13794BP
수탁일자 : 20190122Date of Deposit: 20190122
기탁기관주소: (56212) 대한민국 전라북도 정읍시 입신길 181 한국생명공학연구원(KRIBB)Depository address: (56212) Korea Research Institute of Bioscience and Biotechnology (KRIBB), 181 Ipsin-gil, Jeongeup-si, Jeollabuk-do, Korea
Figure PCTKR2020001222-appb-img-000001
Figure PCTKR2020001222-appb-img-000001
본 발명에 따른 신규한 효모는 Treg이 매개하는 Th2 면역반응을 촉진하여 항염증 활성을 가지며, 정장 효과를 가지고, 오브알부민 감작 마우스 모델에 대하여 피부 및 장에서 염증을 억제하는 효과가 탁월하다. 뿐만 아니라, 내산성 및 내담즙성 또한 우수하여 산업상 이용가능성이 높다. The novel yeast according to the present invention promotes a Th2 immune response mediated by Treg, has anti-inflammatory activity, has a formal effect, and has an excellent effect of suppressing inflammation in the skin and intestine against the ovalbumin-sensitized mouse model. In addition, it has excellent acid resistance and bile resistance, and thus has high industrial applicability.

Claims (20)

  1. 카자츠타니아 투리센시스(kazachstania turicensis) CAU Y1706(수탁번호:KCTC13794BP) 균주.Kazachstania turicensis CAU Y1706 (Accession No.: KCTC13794BP) strain.
  2. 제1항에 있어서, 상기 카자츠타니아 투리센시스는 항염증 활성을 갖는 것을 특징으로 하는 균주. The strain according to claim 1, wherein the Kazaztania thuricensis has anti-inflammatory activity.
  3. 제1항에 있어서, 상기 카자츠타니아 투리센시스는 내산성 또는 내담즙성을 갖는 것을 특징으로 하는 균주.The strain according to claim 1, wherein the Kazaztania thuricensis has acid resistance or bile resistance.
  4. 제1항에 있어서, 상기 카자츠타니아 투리센시스는 Th1/Th2 면역반응 불균형을 개선하는 것을 특징으로 하는 균주.The strain according to claim 1, wherein the Kazaztania thuricensis improves a Th1/Th2 immune response imbalance.
  5. 제1항의 균주 또는 이의 배양물을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising the strain of claim 1 or a culture thereof as an active ingredient.
  6. 제5항에 있어서, 상기 염증성 질환은 급성 또는 만성 장기이식 거부반응, 이식편대숙주병, 염증성 장 질환, 크론병, 궤양성 대장염, 염증성 피부 질환, 다발성 경화증, 췌장염, 외상 유발 쇼크, 기관지 천식, 알러지성 비염, 알러지성 결막염, 낭포성 섬유증, 급성 기관지염, 만성 기관지염, 급성 세기관지염, 만성 세기관지염, 골관절염, 통풍, 척추관절병증, 강직성 척추염, 라이터 증후군, 건선성 관절병증, 장질환 척추염, 연소자성 관절병증, 연소자성 강직성 척추염, 반응성 관절병증, 감염성 관절염, 후-감염성 관절염, 루게릭병, 결절성 다발동맥염, 과민성 혈관염, 루게릭 육아종증, 류마티스성 다발성 근육통, 관절세포 동맥염, 칼슘 결정침착관절병증, 가성 통풍, 비-관절 류마티즘, 점액낭염, 건초염, 상과염, 신경병증성 관절질환(neuropathic joint disease 또는 charcot joint), 출혈성 관절증(hemarthrosis), 알레르기 자반병, 비후성 골관절병증, 다중심성 세망조직구종, 척추측만증(scoliosis), 혈색소증, 혈색소병증, 고지단백혈증, 저감마글로불린혈증, 가족성 지중해열(familial Mediterranean fever), 베체트씨병, 전신성 홍반성 루푸스, 재귀열, 패혈증, 패혈성 쇼크, 급성 호흡곤란 증후군, 다발성 장기부전, 만성 폐쇄성 폐질환(chronic obstructive pulmonary disease), 류마티스성 관절염(rheumatoid arthritis), 급성 폐손상(acute lung injury), 기관지 폐 형성장애(broncho-pulmonary dysplasia), 제1형 당뇨병, 제2형 당뇨병, 동맥경화, 알츠하이머성 치매, 가족성 한랭자가염증 증훈군(familial cold autoinflammatory syndrome), 머클-웰스 증후군(Muckle-Wells syndrome), 신생아발병 다발성 염증성 질환(neonatal multisystem inflammatory disease), 만성 유아 신경피부관절 증후군(chronic infantile neurologic cutaneous articular syndrome), 성인발증형 스틸병(adult-onset Still's disease), 접촉성 피부염, 포상기태(hydatidiform mole), PAPA 증후군(syndrome of pyogenic arthritis, pyoderma gangrenosum and acne), 고면역글로불린 D 증후군(hyperimmunoglobulin d syndrome), 크리오피린 관련 주기적 증후군(cryopyrin-associated periodic syndrome), 각막염, 결막염, 망막염, 망막혈관염, 포도막염, 안검염, 알레르기결막염, 건성 안(dry eye), 전신성 경피증(progressive systemic sclerosis), 다발성 근염(polymyositis), 자가면역성 뇌척수염, 중증 근무력증(myasthenia gravis), 결절성 다발성 동맥염(polyarteritis nodosa) 및 섬유조직염(fibromyalgia syndrome)로 이루어진 군에서 선택되는 어느 하나인 것을 특징으로 하는 조성물.According to claim 5, The inflammatory disease is acute or chronic organ transplant rejection, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, inflammatory skin disease, multiple sclerosis, pancreatitis, trauma-induced shock, bronchial asthma, Allergic rhinitis, allergic conjunctivitis, cystic fibrosis, acute bronchitis, chronic bronchitis, acute bronchiolitis, chronic bronchiolitis, osteoarthritis, gout, spinal joint disease, ankylosing spondylitis, Reiter's syndrome, psoriatic arthrosis, enteropathic spondylitis, combustible joint Pathology, combustive ankylosing spondylitis, reactive arthritis, infectious arthritis, post-infectious arthritis, Lou Gehrig's disease, nodular polyarteritis, irritable vasculitis, Lou Gehrig's granulomatosis, rheumatoid multiple myalgia, articular cell arteritis, calcium degenerative arthrosis, pseudo gout , Non-joint rheumatism, myxitis, hayitis, epicondylitis, neuropathic joint disease or charcot joint, hemorrhagic disease, allergic purpura, hypertrophic osteoarthritis, multi-centre reticulum, scoliosis ), hemochromatosis, hemoglobinemia, hyperlipoproteinemia, hypomaglobulinemia, familial Mediterranean fever, Behcet's disease, systemic lupus erythematosus, recurrent fever, sepsis, septic shock, acute respiratory distress syndrome, multiple organ failure , Chronic obstructive pulmonary disease, rheumatoid arthritis, acute lung injury, broncho-pulmonary dysplasia, type 1 diabetes, type 2 diabetes, Atherosclerosis, Alzheimer's dementia, familial cold autoinflammatory syndrome, Muckle-Wells syndrome, neonatal multisystem inflammatory diseas e), chronic infantile neurologic cutaneous articular syndrome, adult-onset still's disease, contact dermatitis, hydatidiform mole, syndrome of pyogenic arthritis, PAPA syndrome, pyoderma gangrenosum and acne), hyperimmunoglobulin d syndrome, cryopyrin-associated periodic syndrome, keratitis, conjunctivitis, retinitis, retinitis vasculitis, uveitis, blepharitis, allergic conjunctivitis, dry eye eye), systemic sclerosis, polymyositis, autoimmune encephalomyelitis, myasthenia gravis, polyarteritis nodosa, and fibromyalgia syndrome It is characterized in that the composition.
  7. 제6항에 있어서, 상기 염증성 피부 질환은 건선, 아토피성 피부염, 습진성 피부염, 접촉성 피부염, 지루성 피부염, 장미색 비강진, 편평 태선, 혈관염, 모공성 홍색 비강진, 봉와직염, 모낭염, 옹종, 천포창, 수포성(bullous) 천포창, 표피 수포증, 담마진, 혈관 부종, 맥관염, 홍반 및 피부 호산구 증가증으로 이루어진 군에서 선택되는 어느 하나인 것을 특징으로 하는 조성물.The method of claim 6, wherein the inflammatory skin disease is psoriasis, atopic dermatitis, eczematous dermatitis, contact dermatitis, seborrheic dermatitis, rosaceous nasal rash, lichen planus, vasculitis, porous red nasal rash, cellulitis, folliculitis, carcinoma, thrush, can A composition characterized in that it is any one selected from the group consisting of bullous celestial vesicles, epidermal vesicles, gallbladder, angioedema, vasculitis, erythema and skin eosinophilia.
  8. 제1항의 균주 또는 이의 배양물을 유효성분으로 포함하는 염증성 질환의 예방 또는 개선용 식품 조성물.A food composition for preventing or improving an inflammatory disease comprising the strain of claim 1 or a culture thereof as an active ingredient.
  9. 제1항의 균주 또는 이의 배양물을 유효성분으로 포함하는 염증성 질환의 예방 또는 개선용 화장료 조성물.A cosmetic composition for preventing or improving an inflammatory disease comprising the strain of claim 1 or a culture thereof as an active ingredient.
  10. 제1항의 균주 또는 이의 배양물을 유효성분으로 포함하는 생균제 조성물.A probiotic composition comprising the strain of claim 1 or a culture thereof as an active ingredient.
  11. 제1항의 균주 또는 이의 배양물을 유효성분으로 포함하는 정장용 조성물.A dressing composition comprising the strain of claim 1 or a culture thereof as an active ingredient.
  12. 제1항의 균주 또는 이의 배양물을 유효성분으로 포함하는 면역조절용 조성물.A composition for immunomodulation comprising the strain of claim 1 or a culture thereof as an active ingredient.
  13. 제1항의 균주 또는 이의 배양물을 유효성분으로 포함하는 식품첨가용 조성물.A composition for food additives comprising the strain of claim 1 or a culture thereof as an active ingredient.
  14. 염증성 질환의 예방 또는 치료용 제제를 제조하기 위한 제1항의 균주 또는 이의 배양물의 용도.Use of the strain of claim 1 or a culture thereof for preparing a preparation for the prevention or treatment of inflammatory diseases.
  15. 제1항의 균주 또는 이의 배양물을 유효성분으로 포함하는 조성물의 유효량을 이를 필요로 하는 개체에 투여하는 것을 포함하는 염증성 질환의 치료 방법.A method of treating an inflammatory disease, comprising administering an effective amount of a composition comprising the strain of claim 1 or a culture thereof as an active ingredient to an individual in need thereof.
  16. 생균제를 제조하기 위한 제1항의 균주 또는 이의 배양물의 용도.Use of the strain of claim 1 or a culture thereof for producing a probiotic.
  17. 정장용 제제를 제조하기 위한 제1항의 균주 또는 이의 배양물의 용도.Use of the strain of claim 1 or a culture thereof for preparing a formulation for dressing.
  18. 면역조절용 제제를 제조하기 위한 제1항의 균주 또는 이의 배양물의 용도.Use of the strain of claim 1 or a culture thereof for preparing an immunomodulatory agent.
  19. 제1항의 균주 또는 이의 배양물을 유효성분으로 포함하는 조성물의 유효량을 이를 필요로 하는 개체에 투여하는 것을 포함하는 면역조절의 방법.A method of immunomodulation comprising administering an effective amount of a composition comprising the strain of claim 1 or a culture thereof as an active ingredient to an individual in need thereof.
  20. 식품첨가용 제제를 제조하기 위한 제1항의 균주 또는 이의 배양물의 용도.Use of the strain of claim 1 or a culture thereof for preparing a food additive preparation.
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